CN105316415B - The gene quick screening method of four kinds of black yeast bacterium in beer brewing is identified simultaneously - Google Patents
The gene quick screening method of four kinds of black yeast bacterium in beer brewing is identified simultaneously Download PDFInfo
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Abstract
The present invention discloses gene quick screening method that is a kind of while identifying four kinds of black yeast bacterium in beer brewing.This method utilizes the primer system being made of 4 pairs of primers, by using the mode that high-throughput multiple PCR technique is combined with high-resolution capillary electrophoresis separation technology, the detection that whether there is black yeast bacterium in brewer's yeast and fermentation process is quickly determined to beer production to realize.The present invention is relative to other molecular Biological Detection means based on round pcr at present, 4 kinds of beer black yeast bacterium Pichia fermentans of detection can be integrated in primary first-order equation, Candida mesenterica, Hansenula polymorpha, Rhodotorula glutinis, testing cost has been saved, detection time is saved, detection time shortens to 8h in 7~14 days by original.
Description
Technical field
The invention belongs to beer technical fields, are related to gene that is a kind of while identifying four kinds of black yeast bacterium in beer brewing
Quick screening method, more particularly to it is a kind of to be based on multiple PCR technique and capillary electrophoresis separation technology, in beer brewing
The method that several black yeast bacterium are quickly screened.
Background technology
Beer can be divided into two major classes, i.e. Ai Er (ale) beer and glug according to the difference of zymotechnique and strain used
(lager) beer.The former is sent out at a relatively high temperature using S. cervisiae (Saccharomyces cerevisiae)
Ferment, saccharomycete often float on upper layer, therefore also known as high fermentation beer.The latter is fermented under the conditions of low temperature (10 DEG C or so), yeast
Bacterium is often sunken to bottom, therefore also known as lower layer's fermentation beer.Glug beer fermentation strain is known as saccharomyces pastorianus
(Saccharomyces pastorianus)。
Brewing is built upon on the basis of fermentation and control harmful microbe pollution using brewer's yeast.We
It is desirable that the pure-blood ferment that only S. cervisiae participates in, but in entire Process of Beer Brewing, the guarantor of saccharomyces cerevisiae strain
It deposits, the factors such as brewing equipment, air, brewing water are likely to that harmful microorganism is caused to enter in wheat juice or zymotic fluid.
From the point of view of wine brewing process upstream, the high-quality and purity of brewer's yeast is the most key, if being used primarily for the ferment of beer fermentation
Maternal body contains contaminated bacteria (being usually other black yeast bacterium), can be to the beer quality and beer of entire or even several production batch
Wine entirety special favor generates grievous injury, such as beer is rancid, foreign odor.In addition, other black yeast bacterium during the fermentation
Into wheat juice or zymotic fluid, wheat juice and beer can be also polluted.Black yeast bacterium can be in liquor-making environment and S. cervisiae one
Sample, mass propagation amplification.Therefore, initial in brewing, and in brewing process, the purity of S. cervisiae is monitored,
Seem most important.
Black yeast bacterium mainly has Pichia fermentans, Candida mesenterica, Hansenula
Polymorpha, Rhodotorula glutinis.Two classes are broadly divided into for the detection mode of beer black yeast bacterium, first
Class is the culture-based method based on culture medium, and domestic and international most of breweries use such method, but detection time is long；It makes simultaneously
Waste mash is dense, wherein micro black yeast bacterium is cultivated by culture medium, is often difficult to find, therefore can not
Timely Instructing manufacture technical staff is monitored beer production；Second class is the molecular biology inspection of based on PCR technology
Survey method, round pcr greatly improve detection efficiency and sensitivity from nucleic acid, gene level, can be qualitative to kind, category or one
Class object bacteria, detection is quick and qualitative ability is strong, thus no matter monitoring for finished beer quality or process problems
Investigation of tracing to the source, the application of round pcr all has broad prospects and profound significance, but primary first-order equation can only detect it is a kind of micro-
Biology, and exist from a large amount of sensitivity problems for making detection micropollution saccharomycete in waste mash.
Many experiments it has proven convenient that black yeast bacterium Pichia fermentans, Candida mesenterica,
Hansenula polymorpha, Rhodotorula glutinis, discriminating are a more complex job, these black yeasts
Bacterium makes wine uses yeast fungus usually in the form of more micro, with high concentration and mixes, and detection process is complicated and is easy missing inspection, thus has
Necessity carries out more rapidly more sensitive detection method.In order to monitor the microbial status of beer production more in time, having must
Develop it is a kind of for common beer black yeast bacterium speed faster, the better detection method of effect.
Invention content
The purpose of the present invention is in view of the drawbacks of the prior art, overcome black yeast bacterium Pichia in conventional beer
Fermentans, Candida mesenterica, Hansenula polymorpha, Rhodotorula glutinis detections
The deficiencies of technical operation is cumbersome, time-consuming effort provides a kind of while identifying that the gene of four kinds of black yeast bacterium in beer brewing is fast
Fast screening method, using the primer system being made of 4 pairs of primers, by using high-throughput multiple PCR technique and high-resolution
The mode that is combined of capillary electrophoresis separation technology, brewer's yeast and fermentation are quickly determined to beer production to realize
It whether there is the detection of black yeast bacterium in the process.
The technical scheme is that：
The present invention analyzes sample 4 kinds of black yeast bacterium of needle, if any of which contaminated bacteria can be detected, says
Contain the brewery sludge with beer pollution capacity in bright sample.
A kind of quick screening method of beer black yeast bacterium expands the gene of 4 kinds of contaminated bacterias using 4 weight PCR simultaneously, then
By described in capillary electrophoresis separation 4 heavy pcr amplification products, NO.1~8 primer SEQ ID are applied in the 4 weight PCR reactions.
The method of the present invention specific steps include as follows：
Step (1), the extraction of beer black yeast bacterium DNA profiling：
Using general DNA extraction method and purification process, from wine brewing with taking sample in high yeast concentration bacterium, or from wine brewing
Sample is taken in wheat juice or zymotic fluid in the process, extracts to obtain DNA.
Step (2), multiplexed PCR amplification：
After multiplexed PCR amplification reaction system is vibrated mixing, multiplexed PCR amplification is carried out, amplified production is obtained；
The multiplexed PCR amplification reaction system is that 20ul by 100nM forward direction mix primers 2ul, 100nM, reversely draw by mixing
The MgCl of object 2ul, 25mM2The Taq polymerase 0.7ul of 4ul, 5 × PCR Buffer4ul, 5U/ul, DNA profiling 1ul and surplus
Distilled water；SEQ ID NO.1 primers, SEQ ID NO.3 primers, SEQ ID NO.5, SEQ ID in wherein positive mix primer
The concentration ratio of NO.7 primers is 1:1:1:1, SEQ ID NO.2 primers, SEQ ID NO.4 primers, SEQ in reversed mix primer
ID NO.6, SEQ ID NO.8 primers concentration ratio be 1:1:1:1；
The condition of the multiplexed PCR amplification reaction is 95 DEG C of pre-degeneration 10min；With 94 DEG C of 30s；58℃30s；70℃
1min is 1 cycle, carries out 35 cycles altogether；4 DEG C of heat preservations；
4 each primer sequences of weight primer system of the genetic test for black yeast bacterium that 1 present invention of table designs
Step (3), capillary electrophoresis separation condition：
Separation system：The amplified production of gained and volume content are 95% deionized formamide in step described in 1ul (2)
38.5ul, 400bpDNA Marker0.5ul are uniformly mixed；Separation condition：Under 50 DEG C, 6.0KV voltages, 35min is detached；
Step (4), Testing and appraisal：
When the amplified production of gained in the step (2) is passed through capillary electrophoresis separation, lost by multiple gene expression
Pass analysis system automatically retrieval, collect fluorescence signal in sample and judge whether genetic fragment length be 142bp, 147bp,
There is characteristic peak in the corresponding position 335bp, 359bp；If there is characteristic peak in the corresponding positions 142bp, it is judged as the detection
Contain black yeast bacterium Pichia fermentans in sample；If there is characteristic peak in the corresponding positions 147bp, it is judged as
Contain black yeast bacterium Candida mesenterica in the detection sample；If there is characteristic peak in the corresponding positions 335bp,
Then it is judged as containing black yeast bacterium Hansenula polymorpha in the detection sample；If going out in the corresponding positions 359bp
Existing characteristic peak is then judged as containing black yeast bacterium Rhodotorula glutinis in the detection sample.
2 four kinds of black yeast bacterium of table
The present invention has following features compared with prior art：
First, the present invention can carry out in same PCR pipe, and it is easy to operate, it can quickly determine whether there is beer pollution ferment
Female bacterium, and testing result is accurate；
Second, determining beer black yeast bacterium Pichia ermentans, Candida by analyzing gene
Mesenterica, Hansenula polymorpha, Rhodotorula glutinis brewing initially and in the process
Micro presence finds to be harmful to saccharomycete in beer in time, realizes effective monitoring and prevention effect；
Third, the present invention is relative to other molecular Biological Detection means based on round pcr at present, it can be one
4 kinds of beer black yeast bacterium of detection are integrated in secondary response, have saved testing cost, save detection time, detection time is by original 7
Shorten to 8h within~14 days.
Description of the drawings
Fig. 1 is multiplex PCR-Capillary Electrophoresis spectrogram of the specific embodiment of the invention 1；
Fig. 2 is multiplex PCR-Capillary Electrophoresis spectrogram of the specific embodiment of the invention 2.
Specific implementation mode
With reference to specific embodiment, the present invention is further analyzed.
Embodiment 1：
(1) culture of beer black yeast bacterium and DNA profiling extraction：
Beer black yeast bacterium the Pichia ermentans, Candida grown on picking plating medium
Mesenterica, Hansenula polymorpha, Rhodotorula glutinis single bacterium colonies, according to Qiagen companies
The DNA profiling of the operation instruction extraction beer black yeast bacterium of Genomic DNA Purification Kit kits, and make
The DNA mass for measuring extraction under 260nm wavelength conditions with ultraviolet specrophotometer, 250ng/ul is diluted to by DNA solution ,-
20 DEG C save backup.
(2) Quadruple- PCR expands
The primer system is SEQ ID NO.1-8, the 20ulPCR reaction systems described in table 1：The mixing of 100nM forward directions is drawn
Object 2ul, 100nM reversed mix primer 2ul, the MgCl of 25mM2The Taq polymerase of 4ul, 5 × PCRBuffer4ul, 5U/ul
0.7ul, DNA profiling 1ul, supplying distilled water makes total volume be 20ul；95 DEG C of pre-degeneration 10min；With 94 DEG C of 30s；58℃30s；
70 DEG C of 1min are 1 cycle, carry out 35 cycles altogether；4 DEG C of heat preservations；
(3) capillary electrophoresis separation condition
Separation system：The amplified production and 95% deionized formamide 38.5ul of gained in step described in 1ul (2),
400bpDNA Marker0.5ul are uniformly mixed；
Separation condition：Under 50 DEG C, 6.0KV voltages, 35min is detached；
(4) data analysis
It is automatically right using the GenomeLabGeXP Genetic Analysis System analysis systems of Beckman Kurt
Separating resulting is analyzed, and shown in result figure 1, the gene of 4 kinds of black yeast bacterium is detected, using the present invention primary anti-
It answers, the characteristic peak of 4 kinds of black yeast bacterium genes in sample can be captured.
Embodiment 2：
The two major classes of beer, i.e. Ai Er (ale) beer and glug (lager) beer, the former uses S. cervisiae
(Saccharomyces cerevisiae), glug beer fermentation strain are saccharomyces pastorianus (Saccharomyces
Pastorianus), in the upstream of wine brewing process, exist with high concentration.This example can prove the present invention to black yeast bacterium
Pichia fermentans, Candida mesenterica, Hansenula polymorpha, Rhodotorula
The detection specificity of glutinis, i.e., detection method of the invention is in only S. cervisiae (Saccharomyces
Cerevisiae) exist or in the case of saccharomyces pastorianus (Saccharomyces pastorianus) exists, do not show feature
Peak, 2 kinds of non-polluting common saccharomycete (Saccharomyces cerevisiae and Saccharomyces pastorianus) are deposited
Interference will not generated to detection.
The operating procedure of the present invention is as described in Example 1.
(1) culture of brewery sludge and DNA profiling extraction：
The S. cervisiae (Saccharomyces cerevisiae) grown on picking plating medium exists and Bath
Moral yeast (Saccharomyces pastorianus) single bacterium colony, according to the Genomic DNA of Qiagen companies
The DNA profiling of operation instruction the extraction S. cervisiae and saccharomyces pastorianus of Purification Kit kits, and using purple
Outer spectrophotometer measures the DNA mass of extraction under 260nm wavelength conditions, and DNA solution is diluted to 250ng/ul, -20 DEG C
It saves backup.
(2) Quadruple- PCR expands
The primer system is SEQ ID NO.1-8, the 20ulPCR reaction systems described in table 1：The mixing of 100nM forward directions is drawn
Object 2ul, 100nM reversed mix primer 2ul, the MgCl of 25mM2The Taq polymerase of 4ul, 5 × PCRBuffer4ul, 5U/ul
0.7ul, DNA profiling 1ul, supplying distilled water makes total volume be 20ul；95 DEG C of pre-degeneration 10min；With 94 DEG C of 30s；58℃30s；
70 DEG C of 1min are 1 cycle, carry out 35 cycles altogether；4 DEG C of heat preservations；
(3) capillary electrophoresis separation condition
Separation system：The amplified production and 95% deionized formamide 38.5ul of gained in step described in 1ul (2),
400bpDNA Marker0.5ul are uniformly mixed；
Separation condition：Under 50 DEG C, 6.0KV voltages, 35min is detached；
(4) data analysis
It is automatically right using the GenomeLabGeXP Genetic Analysis System analysis systems of Beckman Kurt
Separating resulting is analyzed, the results show that occurring without characteristic peak, i.e. S. cervisiae (Saccharomyces
Cerevisiae) or the presence of saccharomyces pastorianus (Saccharomyces pastorianus) does not influence testing result, no
It will appear false positive results.
Embodiment 3：
In the process of wine brewing, non-polluting fermented yeast bacterium (Saccharomyces cerevisiae and
Saccharomyces pastorianus) fermenting microbe exists with high concentration, black yeast bacterium Pichia fermentans,
Candida mesenterica, Hansenula polymorpha, Rhodotorula glutinis exist with low concentration.This
When example can prove that micro black yeast bacterium and high gravity fermentation strain coexists in the present invention, the present invention is to detecting micro dirt
Contaminate the sensibility of saccharomycete.
This example using 2 kinds of brewing non-polluting common saccharomycete (Saccharomyces cerevisiae and
Saccharomyces pastorianus), with 100,000 times：1 concentration ratio and black yeast bacterium are mixed into sample, to detect this
The sensitivity of invention.
The S. cervisiae (Saccharomyces cerevisiae) grown on picking plating medium respectively and Bath
Moral yeast (Saccharomyces pastorianus) single bacterium colony is added in 100mL culture mediums, the culture extremely system in aerobic environment
At bacteria suspension, spectrophotometric OD=0.2 or so.Black yeast bacterium Pichia fermentans, Candida mesenterica,
Hansenula polymorpha, Rhodotorula glutinis single bacterium colonies are added in 5mL culture mediums, are trained in aerobic environment
It supports to bacteria suspension, spectrophotometric OD=0.2 or so is made.Bacteria suspension concentration is calculated according to OD values, with 100,000 times：1 concentration, will
S. cervisiae or saccharomyces pastorianus and black yeast bacterium mixing.The 1mL bacteria suspensions are taken to be added in 1.5ml centrifuge tubes, 13,000
× g centrifuges 2min, collects cell precipitation, removes supernatant；Cell is thoroughly resuspended in the 50mMEDTA that 480ul is added；120ul is added
10mg/ml lyase bacterium, 37 DEG C incubation 60min, 13,000 × g centrifuge 2min, remove supernatant.
It is extracted later according to the operation instruction of the Genomic DNA Purification Kit kits of Qiagen companies
DNA profiling, multi-PRC reaction system use Thermo-DNA Polymerase DNA kits, use 4 heavy PCR reactants
System, the primer system are SEQ ID NO.1-8, the 20ulPCR reaction systems described in table 1：100nM forward direction mix primer 2ul,
The Taq polymerase 0.7ul, DNA of 100nM reversed mix primer 2ul, the MgCl24ul of 25mM, 5 × PCR buffer solutions 4ul, 5U/ul
Template 1ul, supplying distilled water makes total volume be 20ul；95 DEG C of pre-degeneration 10min；With 94 DEG C of 30s；58℃30s；70 DEG C of 1min are
1 cycle carries out 35 cycles altogether；4 DEG C of heat preservations；Capillary electrophoresis separation condition：Using separation system：The above-mentioned multiplex PCRs of 1ul
Amplified production and 95% deionized formamide 38.5ul, 400bpDNA Marker0.5ul are uniformly mixed；Separation condition：50
DEG C, under 6.0KV voltages, detach 35min, use the GenomeLabGeXP Genetic Analysis of Beckman Kurt
System analysis systems automatically analyze separating resulting, obtain multiplex PCR-Capillary Electrophoresis spectrogram, with 100,000 times it is dense
The S. cervisiae (Saccharomyces cerevisiae) of degree or saccharomyces pastorianus (Saccharomyces
Pastorianus) under Coexistence Situation, black yeast bacterium Pichia fermentans, Candida are capable of detecting when
The characteristic peak of mesenterica, Hansenula polymorpha, Rhodotorula glutinis genes, the results show that 10
The S. cervisiae (Saccharomyces cerevisiae) of ten thousand times of concentration mixes, Neng Goujian with Pichia fermentans
The gene expression characteristics peak of Pichia fermentans is surveyed, it is shown to illustrate to pollute detection containing a large amount of non-polluting saccharomycete in sample
The sensitivity of saccharomycete does not interfere, and shows high sensitivity.
Sequence table
Above-described embodiment is not for the limitation of the present invention, and the present invention is not limited only to above-described embodiment, as long as meeting
The present invention claims all belong to the scope of protection of the present invention.
SEQUENCE LISTING
<110>Electronic University Of Science & Technology Of Hangzhou
<120>The gene quick screening method of four kinds of black yeast bacterium in beer brewing is identified simultaneously
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<170> PatentIn version 3.3
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Claims (4)
1. identifying the gene quick screening methods of four kinds of black yeast bacterium in beer brewing simultaneously, it is characterised in that this method includes
Following steps：
Step (1), the extraction of beer black yeast bacterium DNA profiling：
Using general DNA extraction method and purification process, from wine brewing with taking sample in high yeast concentration bacterium, or from wine brewing process
Sample is taken in middle wheat juice or zymotic fluid, extracts to obtain DNA；
Step (2), multiplexed PCR amplification：
After multiplexed PCR amplification reaction system is vibrated mixing, multiplexed PCR amplification is carried out, amplified production is obtained；
The multiplexed PCR amplification reaction system is 20 μ l, is reversely mixed by 2 μ L of 100nM forward directions mix primer, 100nM
Close the MgCl of primer 2 μ L, 25mM20.7 μ of Taq polymerase of 4 μ L, 5 × PCR Buffer, 4 μ L, 5U/ μ L
L, 1 μ l of DNA profiling and surplus distilled water；SEQ ID NO.1 primers, SEQ ID NO.3 draw in wherein positive mix primer
Object, SEQ ID NO.5 primers, SEQ ID NO.7 primers concentration ratio be 1:1:1:1, SEQ ID NO.2 in reversed mix primer
Primer, SEQ ID NO.4 primers, SEQ ID NO.6 primers, SEQ ID NO.8 primers concentration ratio be 1:1:1:1；
Step (3), capillary electrophoresis separation condition；
Step (4), Testing and appraisal：
When the amplified production of gained in the step (2) is passed through capillary electrophoresis separation, pass through multiple gene expression heredity point
Analysis system automatically retrieval, collect sample in fluorescence signal judge whether genetic fragment length be 142bp, 147bp, 335bp,
There is characteristic peak in the corresponding positions 359bp；If there is characteristic peak in the corresponding positions 142bp, it is judged as in the detection sample
Contain black yeast bacterium Pichia fermentans；If there is characteristic peak in the corresponding positions 147bp, it is judged as the detection
Contain black yeast bacterium Candida mesenterica in sample；If there is characteristic peak in the corresponding positions 335bp, judge
To contain black yeast bacterium Hansenula polymorpha in the detection sample；If there is feature in the corresponding positions 359bp
Peak is then judged as containing black yeast bacterium Rhodotorula glutinis in the detection sample.
2. the gene quick screening method as described in claim 1 for identifying four kinds of black yeast bacterium in beer brewing simultaneously,
It is characterized in that the condition of the multiplexed PCR amplification reaction described in step (2) is 95 DEG C of pre-degeneration 10min；With 94 DEG C of 30s；58℃
30s；70 DEG C of 1min are 1 cycle, carry out 35 cycles altogether；4 DEG C of heat preservations.
3. the gene quick screening method as described in claim 1 for identifying four kinds of black yeast bacterium in beer brewing simultaneously,
It is characterized in that step (3) separation system：In step (2) described in 1 μ L the amplified production of gained and volume content be 95% go from
38.5 μ L, 400bp DNA Marker of sub- formamide, 0.5 μ L are uniformly mixed.
4. the gene quick screening method as described in claim 1 for identifying four kinds of black yeast bacterium in beer brewing simultaneously,
It is characterized in that step (3) separation condition：Under 50 DEG C, 6.0KV voltages, 35min is detached.
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