CN108913807A - For screening multi-primers, kit and the screening technique of the yeast strain of low protease A gene expression - Google Patents
For screening multi-primers, kit and the screening technique of the yeast strain of low protease A gene expression Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract
The present invention proposes a kind of for screening multi-primers, kit and the screening technique of the yeast strain of low protease A gene expression, belong to field of food detection, is able to solve traditional yeast strain problem that time-consuming, detection process is cumbersome, detection cycle is long for screening low protease A gene expression by measuring protease A vigor.The technical solution is included the design of multi-primers, is successively carried out reverse transcription synthesis cDNA template, multiplexed PCR amplification reaction, capillary electrophoresis separation using yeast strain total serum IgE as template, and is analyzed using GeXP system capillary electrophoresis separation result；It is control with reference gene, the expression quantity of the calculated by peak area protease A gene at peak is corresponded in conjunction with key gene, and judge yeast strain for low yield protease A bacterial strain, middle production protease A bacterial strain or high proteinase yield A bacterial strain according to the expression quantity of protease A gene.In this way, the low yeast strain of protease A gene expression amount can accurately and rapidly be filtered out.
Description
Technical field
The present invention relates to field of food detection, more particularly, to screen the yeast strain of low protease A gene expression
Multi-primers, kit and screening technique.
Background technique
Pure white fine and smooth and lasting foam is the one of the important signs that of quality beer, and the persistence of foam steeps holding property index
It measures, defines bubble the holding property standard of top grade beer, bottle of liquor >=180 second, tinned wine >=150 second in national standard.Protein is
The framework ingredient for constituting beer foam, steeping the decaying held is mainly caused by being destroyed as protein by protease A.Protease A
From brewer's yeast, will be released when yeast autolysis, gradually the hydrophobic protein in decomposing and fermenting liquid or
The characteristic for changing hydrophobic protein, to influence the holding property of bubble of fermentation liquid.Therefore, the ferment of low protease A gene expression is screened
Mother strains are of great significance to beer fermentation field.
The yeast strain of low protease A gene expression is screened at present mainly using protease A vigor in measurement fermentation liquid
Method realizes that protease A derives from brewer's yeast in fermentation liquid, and vigor size reflects the protease A gene of brewer's yeast
Therefore expression quantity can filter out the yeast strain of low protease A gene expression by this method.But protease A vigor
Detection method time-consuming, detection process is cumbersome.And it needs yeast strain carrying out fermentation test, detection cycle before detection
It is long.
Summary of the invention
The application be directed to by measure protease A vigor screen low protease A gene expression yeast strain time-consuming, examine
The problem that survey process is cumbersome, detection cycle is long, proposes a kind of for screening the more of the yeast strain of low protease A gene expression
Weight primer, kit and screening technique.
In order to achieve the above object, the present invention provides a kind of for screening the yeast strain of low protease A gene expression
Multi-primers, including being separately designed according to the homologous gene PEP4-Sc and PEP4-Sb of yeast proteinase A key gene PEP4
Specific upstream and downstream primer, specifically includes：
The present invention also provides a kind of kits, comprising as described in the above technical scheme for screening low protease A base
Because of the multi-primers of the yeast strain of expression.
Low protease A gene table is screened using multi-primers as described in the above technical scheme the present invention also provides a kind of
The method of the yeast strain reached, includes the following steps：
Reverse transcription synthesis cDNA template, multiplexed PCR amplification reaction, capillary are successively carried out using yeast strain total serum IgE as template
Electrophoresis tube separation, and capillary electrophoresis separation result is analyzed using GeXP system；
It is control with reference gene, the expression quantity of the calculated by peak area protease A gene at peak is corresponded in conjunction with key gene, and
Judge that yeast strain is laid eggs for low yield protease A bacterial strain, middle production protease A bacterial strain or height according to the expression quantity of protease A gene
White enzyme A bacterial strain.
Preferably, the reference gene includes homologous gene ACT1-Sc, ACT1-Sb of ACT1, set according to reference gene
Specific upstream and downstream primer is counted, is specifically included：
Preferably, being control with reference gene, the calculated by peak area protease A gene at peak is corresponded in conjunction with key gene
The specific calculating of expression quantity and judgment method are：
PEP4-Sc gene expression amount=PEP4-Sc peak area/(ACT1-Sc peak area+ACT1-Sb peak area)/2
When protease A gene expression amount≤3, determine that yeast strain is low yield protease A bacterial strain；When 3<Protease A base
Because of expression quantity<When 10, judgement yeast strain is middle production protease A bacterial strain；When protease A gene expression amount >=10, ferment is determined
Mother strains are high proteinase yield A bacterial strain.
Preferably, using yeast strain total serum IgE as template, with sequence SEQ ID NO.2, SEQ ID NO.4, SEQ ID
Downstream primer shown in NO.6 and SEQ ID NO.8 is that specific primer reverse transcription synthesizes the first chain of cDNA template.
Preferably, the response parameter of the reverse transcription synthesis cDNA template is set as：48 DEG C, 1min；42 DEG C, 60min；
95 DEG C, 5min.
Preferably, using the first chain of cDNA template of synthesis as template, with sequence SEQ ID NO.1, SEQ ID NO.3,
Upstream primer shown in SEQ ID NO.5 and SEQ ID NO.7 is that specific primer carries out multiplexed PCR amplification reaction.
Preferably, the parameter of the multiplexed PCR amplification reaction is set as：95 DEG C of initial denaturation 10min；94 DEG C of denaturation 30s；
55 DEG C of annealing temperature, 30s；70 DEG C of extension 1min, entire pcr amplification reaction process recycle 35 times.
Compared with prior art, the advantages and positive effects of the present invention are：
1, by detection yeast proteinase A gene expression amount, it is low protease A gene expression amount can accurately to be filtered out
Yeast strain, and it is easy to operate, quick.
2, using the multi-functional genetic analysis systems of GeXP, yeast proteinase A can be detected quickly, accurately, with high throughput and closed
The expression quantity of key gene has the characteristics that stronger specificity, sensitivity, the degree of automation, ensure that result reliability and
Repeatability.
Detailed description of the invention
Fig. 1 is the multiplex amplification product electrophoresis result of the yeast proteinase A key gene and reference gene in embodiment 1.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
A kind of method of quick detection industry saccharomyces pastorianus protease A gene, extraction, reverse transcription reaction including total serum IgE
Prepare cDNA template, multi-PRC reaction, capillary electrophoresis, product fragment analysis.
(1) multi-primers design
According to industrial saccharomyces pastorianus protease A gene PEP4-Sc, PEP4-Sb and reference gene ACT1-Sc, ACT1-Sb
Full length sequence, design be suitable for GeXP detection specific upstream and downstream multi-primers (table 1).
The specific upstream and downstream primer of the industrial saccharomyces pastorianus protease A gene of table 1 and reference gene
(2) Total RNAs extraction and purifying
1) activated yeast culture
Bacterial strain one ring in inclined-plane is taken to access in the test tube for filling the sterile wheat juice of 5mL with transfer needle, 25 DEG C of cultures are for 24 hours；
3-5 ring is taken to be filled in the test tube of 5mL wheat juice to another, 25 DEG C of cultures are for 24 hours.
2) yeast pre-treatment
Bacteria suspension is centrifuged 2min in 12000rpm；
Abandoning supernatant, every 3 × 108A yeast cells is rapidly added the RNAlater solution of 0.5-1mL；
4 DEG C of refrigerator storage 1h or processing overnight；
12000rpm is centrifuged 2min, abandons supernatant, deposits in -80 DEG C of refrigerators, takes out when extracting RNA.
3) extraction of yeast cells RNA
According to the process of RNA extracts kit, yeast rna is extracted in step-by-step operation.
200 μ L phosphate buffers are added into sample, supernatant is removed in 12000rpm centrifugation.200 μ L phosphate buffer weights are added
It is outstanding to add 20 μ L lywallzymes, 30 DEG C of processing 45min；
600 μ L lysates are added, 10 μ L 2 mercapto ethanols are added in 1mL lysate, 12000rpm room temperature is centrifuged 2min,
Supernatant is shifted into the centrifuge tube of RNase-free；
587 μ L dehydrated alcohols, sufficient vortex is added.Take 500 μ L to pillar, 12000rpm centrifugation 15s；
700 μ L washing buffer I, 12000rpm are added and are centrifuged 15s.Be added 500 μ L washing buffer II, 12000rpm from
Heart 15s；
The RNase-Free water of 80 DEG C of 30 μ L is added, room temperature 12000rpm is centrifuged 2min；
2 μ L DNase I are added in 37 DEG C of processing 40min, 2 μ L DNase deactivators are added in being placed at room temperature for 5min,
12000rpm is centrifuged 2min, the total serum IgE of purifying can be obtained, immediately detectable concentration, and -80 DEG C of refrigerators save；RNA purity testing：
If the OD260/280 of measurement is between 1.8 and 2.1, it can be used to subsequent experimental, otherwise extract again.
(3) reverse transcription reaction prepares cDNA template
Using yeast total serum IgE as the first chain of templated synthesis cDNA, the downstream primer of multi-primers is specific primer in table 1.
It is as follows that reverse transcription reaction prepares cDNA template reaction parameter setting：
48 DEG C, 1min；42 DEG C, 60min；95 DEG C, 5min.
(4) multi-PRC reaction
Using the first chain of cDNA of above-mentioned synthesis as template, the upstream primer of multi-primers is specific primer in table 1, is carried out
Multiplexed PCR amplification reaction.
PCR amplification parameter setting is as follows：
95 DEG C of initial denaturation 10min；94 DEG C of denaturation 30s；55 DEG C of annealing temperature, 30s；70 DEG C of extension 1min, entire PCR amplification
Reaction process recycles 35 times.
(5) multiple PCR products Capillary Electrophoresis
1 μ L PCR multiple products are taken to be added to 95% deionized formamide (SLS) and 400bp Marker that packing there are 39 μ L
A drop paraffin oil is covered in the hole of the upper template of mixed liquor, after being mixed with liquid-transfering gun.In addition 250 are added in the every hole of buffer plate
The dissociating buffer of μ L.After all having prepared, upper machine carries out Capillary Electrophoresis.Separation gel, dissociating buffer are purchased from Beckman
Coulter。
(6) product fragment analysis
The detection of protease A gene expression amount is carried out according to the testing process of the multi-functional genetic analysis systems of GeXP：First
Capillary electrophoresis separation is carried out, the result is shown in Figure 1 analyzes Capillary Electrophoresis result using GeXP system parameter, record knot
Fruit.Each peak indicates that corresponding gene, the area at each peak correspond to the expression quantity of gene, wherein the peak area knot at each peak
Fruit is shown in Table 2.Its corresponding peak area is searched according to protease A gene size, reference gene size, system control size.System
Whether the peak figure of control represents Capillary Electrophoresis result normal.
2 protease A key gene of table and the corresponding peak area result of reference gene
| Serial number | Gene Name | Peak area |
| 1 | PEP4-Sc | 37063 |
| 2 | PEP4-Sb | 7946 |
| 3 | ACT1-Sc | 3702 |
| 4 | ACT1-Sb | 1293 |
(7) it is control with reference gene, calculates the expression quantity of protease A gene.
PEP4-Sc gene expression amount=PEP4-Sc peak area/(ACT1-Sc peak area+ACT1-Sb peak area)/2
PEP4-Sb gene expression amount=PEP4-Sb peak area/(ACT1-Sc peak area+ACT1-Sb peak area)/2
It is 3.18 that PEP4-Sc gene expression amount is calculated, which is 14.84, PEP4-Sb gene expression amount, due to the source Sc
Protease A gene expression amount is much higher than the gene expression amount in the source Sb, and the fluctuation of its expression quantity at different conditions is larger, by
External environment influences greatly, to show that the protease A gene in the source Sc plays a leading role in yeast strain, therefore according to the source Sc
Protease A gene expression amount screens yeast strain, judges that the yeast strain produces protease A bacterial strain for X.
Embodiment 2
Tri- different yeast strains of A, B, C are taken, the measuring method of protease A gene expression amount is the same as embodiment 1.
Three yeast strains are spread cultivation step by step respectively, the brewing for 100L beer is tested.In addition to bacterial strain, production technology ginseng
Number is all consistent.
(1) yeast spread cultivation process
It is accessed in the test tube for filling the sterile wheat juice of 5mL, is shaken up, 25 DEG C from one ring of picking in preservation inclined-plane bacterial strain with transfer needle
Activation culture is for 24 hours；It draws 0.5mL bacteria suspension to be linked into the test tube that another fills the sterile wheat juice of 5mL, shake up, 25 DEG C of activation
Culture is for 24 hours；It draws 1mL bacteria suspension to be linked into the test tube for filling the sterile wheat juice of 10mL, shake up, 25 DEG C of activation cultures are for 24 hours；It will
10mL bacteria suspension is linked into the triangular flask for filling the sterile wheat juice of 200mL, is shaken up, and 22 DEG C of activation cultures are for 24 hours；200mL bacterium is hanged
Liquid is linked into the triangular flask for filling the sterile wheat juice of 2L, is shaken up, and 19 DEG C of activation cultures are for 24 hours；2L bacteria suspension is linked into and fills 20L
It in the Cattell tank of sterile wheat juice, shakes up, 16 DEG C of activation cultures are for 24 hours；20L bacteria suspension is linked into the fermentor for filling 100L wheat juice
In.
(2) technological condition for fermentation
10 DEG C of canful temperature, 13 ° of P wheat juice canful yeast number 22.0MM/mL, 10 DEG C of main ferment temperature, appearance pol drops to 5 ° of P
When, 12 DEG C are warming up to, yeast is recycled, sealed cans when appearance pol drops to 3.0 DEG C carry out biacetyl reduction, and biacetyl drops to 10 μ g/
Quickly cooling when L, storage wine temperature are 0~-1.5 DEG C, and the storage wine time is 7 days, and the holding property of bubble of cold storage wine is measured after filtering.
(3) interpretation of result
3 different strains testing result of table
| Yeast strain | Protease A gene expression amount | Protease A vigor | Cold storage wine steeps holding property (second) |
| A | 1.6 | 15 | 225 |
| B | 8.3 | 39 | 203 |
| C | 15.5 | 71 | 167 |
By 3 result of table it can be found that the protease A gene expression amount of A yeast is low, cold holding property of storage wine bubble is higher；C yeast
Protease A gene expression amount it is high, it is lower that cold storage wine steeps holding property.The direct reactive protein enzyme A of protease A gene expression amount is living
Power, therefore can use it and low protease A gene expression yeast strain is quickly screened.Compared to by measuring protease
The vigor of A screens the yeast strain of low protease A gene expression, by detection protease A gene expression amount screens low egg
The yeast strain time-consuming of white enzyme A gene expression is short, as a result accurately, simultaneously as the process uses the multi-functional genetic analysis of GeXP
System, can quickly, expression quantity that is accurate, detecting yeast proteinase A key gene with high throughput, have stronger specificity,
The features such as sensitivity, the degree of automation, ensure that the reliability and repeatability of result.
Sequence table
<110>Qingdao Beer Co., Ltd.
<120>For screening multi-primers, kit and the screening technique of the yeast strain of low protease A gene expression
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 38
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<213>Artificial sequence (Artificial Sequence)
<400> 1
aggtgacact atagaatatg gtcagcgcca accaagtt 38
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<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gtacgactca ctatagggag ggcaacttcg gggttagctt 40
<210> 3
<211> 42
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aggtgacact atagaatacg gactgtaaca ctagagacgg ct 42
<210> 4
<211> 43
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gtacgactca ctatagggag ccccgtaaat ggagtagtat ttg 43
<210> 5
<211> 35
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
aggtgacact atagaataac gctcctcgtg ctgtc 35
<210> 6
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<213>Artificial sequence (Artificial Sequence)
<400> 6
gtacgactca ctatagggat agaaggctgg aacgttaa 38
<210> 7
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<213>Artificial sequence (Artificial Sequence)
<400> 7
aggtgacact atagaatagg catcacacct tttaca 36
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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gtacgactca ctatagggaa ttccgactcg tccaacatc 39
Claims (9)
1. a kind of for screening the multi-primers of the yeast strain of low protease A gene expression, which is characterized in that including according to ferment
The homologous gene PEP4-Sc and PEP4-Sb of female protease A key gene PEP4 separately designs specific upstream and downstream primer, specifically
Including：
2. a kind of kit, which is characterized in that comprising as described in claim 1 for screening low protease A gene expression
The multi-primers of yeast strain.
3. a kind of method for the yeast strain that low protease A gene expression is screened using multi-primers described in claim 1,
It is characterized in that, includes the following steps：
Reverse transcription synthesis cDNA template, multiplexed PCR amplification reaction, capillary electricity are successively carried out using yeast strain total serum IgE as template
Swimming separation, and capillary electrophoresis separation result is analyzed using GeXP system；
It is control with reference gene, the expression quantity of the calculated by peak area protease A gene at peak is corresponded in conjunction with key gene, according to egg
The expression quantity of white enzyme A gene judges yeast strain for low yield protease A bacterial strain, middle production protease A bacterial strain or high proteinase yield A bacterium
Strain.
4. method as claimed in claim 3, which is characterized in that the reference gene include ACT1 homologous gene ACT1-Sc,
ACT1-Sb designs specific upstream and downstream primer according to reference gene, specifically includes：
5. method as claimed in claim 4, which is characterized in that with reference gene to compare, correspond to peak in conjunction with key gene
The specific calculating of the expression quantity of calculated by peak area protease A gene and judgment method are：
PEP4-Sc gene expression amount=PEP4-Sc peak area/(ACT1-Sc peak area+ACT1-Sb peak area)/2
When protease A gene expression amount≤3, determine that yeast strain is low yield protease A bacterial strain；When 3<Protease A gene table
Up to amount<When 10, judgement yeast strain is middle production protease A bacterial strain；When protease A gene expression amount >=10, saccharomycete is determined
Strain is high proteinase yield A bacterial strain.
6. method as claimed in claim 4, which is characterized in that using yeast strain total serum IgE as template, with sequence SEQ ID
NO.2, SEQ ID NO.4, downstream primer shown in SEQ ID NO.6 and SEQ ID NO.8 are specific primer reverse transcription synthesis
The first chain of cDNA template.
7. method as claimed in claim 6, which is characterized in that the response parameter setting of the reverse transcription synthesis cDNA template
For：48 DEG C, 1min；42 DEG C, 60min；95 DEG C, 5min.
8. method as claimed in claim 4, which is characterized in that using the first chain of cDNA template of synthesis as template, with sequence SEQ
It is more that ID NO.1, SEQ ID NO.3, upstream primer shown in SEQ ID NO.5 and SEQ ID NO.7 are that specific primer carries out
Weight pcr amplification reaction.
9. method according to claim 8, which is characterized in that the parameter of the multiplexed PCR amplification reaction is set as：95 DEG C pre-
It is denaturalized 10min；94 DEG C of denaturation 30s；55 DEG C of annealing temperature, 30s；70 DEG C of extension 1min, entire pcr amplification reaction process circulation 35
It is secondary.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104403957A (en) * | 2014-12-16 | 2015-03-11 | 天津科技大学 | Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain |
| CN105385615A (en) * | 2015-12-28 | 2016-03-09 | 天津科技大学 | Saccharomyces cerevisiae strain with high yield of ester and low yield of higher alcohol as well as building and application of saccharomyces cerevisiae strain |
| CN108866027A (en) * | 2014-12-02 | 2018-11-23 | 天津科技大学 | Application of the VPS10 gene in the low secretory protein A of Wine brewing yeast strain |
-
2018
- 2018-08-16 CN CN201810959649.4A patent/CN108913807B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108866027A (en) * | 2014-12-02 | 2018-11-23 | 天津科技大学 | Application of the VPS10 gene in the low secretory protein A of Wine brewing yeast strain |
| CN104403957A (en) * | 2014-12-16 | 2015-03-11 | 天津科技大学 | Saccharomyces cerevisiae strain for low protease A extra-cellular secretion under stress condition and construction method of saccharomyces cerevisiae strain |
| CN105385615A (en) * | 2015-12-28 | 2016-03-09 | 天津科技大学 | Saccharomyces cerevisiae strain with high yield of ester and low yield of higher alcohol as well as building and application of saccharomyces cerevisiae strain |
Non-Patent Citations (2)
| Title |
|---|
| HIROTO KONDO等: "Advanced Method for Measuring Proteinase A in Beer and Application to Brewing", 《JOURNAL OF THE INSTITUTE OF BREWING》 * |
| YANG HE等: "Monitoring of the production of flavor compounds by analysis of the gene transcription involved in higher alcohol and ester formation by the brewer’s yeast Saccharomyces pastorianus using a multiplex RT-qPCR assay", 《INSTITUTE OF BREWING & DISTILLING》 * |
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