CN107641644A - A kind of method for detecting yellow rice wine putrefactive microorganisms - Google Patents
A kind of method for detecting yellow rice wine putrefactive microorganisms Download PDFInfo
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Abstract
The present invention relates to a kind of method for detecting yellow rice wine putrefactive microorganisms, belong to technical field of microbial detection.The present invention devises the specific primer of 5 pairs of lactobacillus acetotoleranses for being used for quantitative fluorescent PCR, and describes a kind of fluorescence quantitative detecting method of yellow rice wine putrefactive microorganisms lactobacillus acetotolerans in detail, and the detection method has good accuracy, repeatability and stability.The forecast analysis that quick detection analysis and yellow rice wine of this method available for microorganism of being become sour in brewing yellow rice wine, storage process are become sour.
Description
Technical field
The present invention relates to a kind of method for detecting yellow rice wine putrefactive microorganisms, belong to technical field of microbial detection.
Background technology
Yellow rice wine is China's wine kind with the longest history, with beer, grape wine and the referred to as great Gu wine of the world three.Yellow rice wine be with rice,
Milled glutinous broomcorn millet, millet, corn, wheat etc. are raw material, with bent class and distiller's yeast etc. for saccharifying ferment, through boiling, diastatic fermentation, pressure
Filter, clarification, sterilization, storage, allotment, filtering, bottling, then the brewed wine that the process such as row sterilization forms.
In the production process of yellow rice wine, the phenomenon of generally existing corruption pollution.Because brewing yellow rice wine is open multi-strain fermentation,
Mainly beneficial microbe is set normally to breed fermentation by the control and operating environment health of process conditions.If in yellow wine fermentation process
In, putrefactive microorganisms excess growth breeding, metabolism produces volatile or non-volatile organic acid, yellow rice wine is become sour；
In altar storage process of making pottery, due to leakage altar, the reason such as unreal is sealed, the growth of yellow rice wine contaminating microorganisms can be caused, cause wine body
Acidity raises or acidity rise and muddiness, it sometimes appear that peculiar smell, different gas, serious is even smelly, due to being stored in Tao Tan
Cheng Zhong, Tao Tan are in sealing state, can not detect the wine body situation of yellow rice wine in real time, so often late when finding.With
The development of the big tank Production trend of yellow rice wine, the influence that yellow rice wine becomes sour to rice wine production is more very important.
Yellow rice wine becomes sour mainly as caused by yellow rice wine putrefactive microorganisms.However, also have one to yellow rice wine putrefactive microorganisms understanding at present
A little defects, main putrefactive microorganisms species are indefinite.Moreover, the discovery for putrefactive microorganisms at present, is all that use can train
Foster mode, required time length be present, yellow wine fermentation or storage process can not be tracked in time so as to take in time targetedly
The problems such as measure.
The content of the invention
In order to solve the above problems, the invention provides a kind of method that detection yellow rice wine, analysis yellow rice wine whether there is corrupt potential quality.
Inventor is separately cultured to have obtained the main putrefactive microorganisms of yellow rice wine, lactobacillus acetotolerans.Lactobacillus acetotolerans has higher alcohol resistance to
By property, the alcoholic strength of ability 21% -25%, the most suitable growth pH is 4.5-5.0, can be grown in yellow rice wine, cause yellow rice wine to become sour.
First purpose of the present invention is to provide a kind of method of quick detection yellow rice wine putrefactive microorganisms, and methods described is to use fluorescence
Quantitative PCR detection lactobacillus acetotolerans.
In one embodiment of the invention, the specific primer used during the fluorescence quantitative PCR detection is to for primer pair
P1 (sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2), P2 (sequence such as SEQ ID NO.3 and SEQ ID NO.4
It is shown), P3 (sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6), P4 (sequence such as SEQ ID NO.7 and SEQ ID
Shown in NO.8) or P5 (sequence is as shown in SEQ ID NO.9 and SEQ ID NO.10) in any pair.
In one embodiment of the invention, the specific primer to for primer pair P1 (sequence such as SEQ ID NO.1 and
Shown in SEQ ID NO.2).
Second object of the present invention is to provide a kind of method for controlling yellow rice wine quality, and methods described is to detect and control in yellow rice wine
The content of lactobacillus acetotolerans.
In one embodiment of the invention, the control yellow rice wine quality refers to prevent yellow rice wine from becoming sour.
In one embodiment of the invention, methods described is that the content for controlling the lactobacillus acetotolerans in yellow rice wine is less than 100
CFU/mL。
In one embodiment of the invention, the detection is to use fluorescence quantitative PCR detection lactobacillus acetotolerans.
In one embodiment of the invention, the specific primer used during the fluorescence quantitative PCR detection is to for primer pair
Any pair in P1, P2, P3, P4 or P5.
In one embodiment of the invention, methods described is when the content for detecting the lactobacillus acetotolerans in yellow rice wine exceedes
During 1.00E+02CFU/mL, take measures to reduce the content of lactobacillus acetotolerans to 100CFU/mL is less than, then proceed at storage
Yellow rice wine after reason continues to store after the yellow rice wine after processing is mixed with other yellow rice wine up to standard.
In one embodiment of the invention, methods described is that the content for controlling the lactobacillus acetotolerans in yellow rice wine is less than 62
CFU/mL。
Third object of the present invention is to provide a kind of method for detecting yellow rice wine, is to detect the lactobacillus acetotolerans in yellow rice wine
(Lactobacillus acetotolerans)。
In one embodiment of the invention, methods described, whether deposited by the presence situation analysis yellow rice wine of lactobacillus acetotolerans
In the danger become sour.
In one embodiment of the invention, methods described, using the presence or absence of fluorescence quantitative PCR detection lactobacillus acetotolerans,
Or the content of lactobacillus acetotolerans.
In one embodiment of the invention, methods described, it is the content of quantitative detection lactobacillus acetotolerans, works as lactobacillus acetotolerans
During more than 1.00E+02CFU/mL, take measures to reduce the content of lactobacillus acetotolerans.
In one embodiment of the invention, it is described to take measures to refer to sterilize or sterilize, it is less than lactobacillus acetotolerans content
100CFU/mL, then proceed to the yellow rice wine after storage processing or continue to store up after the yellow rice wine after processing is mixed with other yellow rice wine up to standard
Deposit.
In one embodiment of the invention, the specific primer used during the fluorescence quantitative PCR detection is to for primer pair
Any pair in P1, P2, P3, P4 or P5.
In one embodiment of the invention, the quantitative fluorescent PCR is SYBR Green I fluorescent quantitations PCR.
In one embodiment of the invention, the reaction system of the quantitative fluorescent PCR is：Sterilized water 8.2ul, SYBR Green
I 10ul, forward primer 0.4ul, reverse primer 0.4ul, DNA profiling 1ul；Quantitative fluorescent PCR response procedures：95 DEG C of pre- changes
Property 5min；95 DEG C of denaturation 10s, 60 DEG C of 30s, collect fluorescence signal, expand 40 circulations；65 DEG C -95 DEG C heatings, are collected
Fluorescence signal, draw melting curve.
Fourth object of the present invention is to provide a species-specific primer pair, the specific primer to be primer pair P1, P2, P3,
Any pair in P4 or P5.
Beneficial effects of the present invention：
(1) present invention firstly discovers that lactobacillus acetotolerans become sour with yellow rice wine it is closely bound up.Can be with by the presence situation of lactobacillus acetotolerans
Danger of the yellow rice wine with the presence or absence of corruption is reacted, and then is easy to take measures to prevent yellow rice wine from becoming sour in time.
(2) present invention develops a kind of method that quick detection yellow rice wine whether there is become sour danger and then progress yellow rice wine quality control,
It is to use real-time fluorescence quantitative PCR.The high specificity of real-time fluorescence quantitative PCR primer of the present invention, real-time fluorescence are fixed
Amount PCR detection method has specific height, high sensitivity, reproducible, stability is good, accuracy is good, pollution-free, consumption
When it is short, easy to be quick the advantages that, can simultaneously realize and to be quantified for lactobacillus acetotolerans in different samples, be become sour for research yellow rice wine
Have great importance.
(3) quick detection analysis and yellow rice wine of the inventive method available for microorganism of being become sour in brewing yellow rice wine, storage process is become sour
Forecast analysis.
Brief description of the drawings
Fig. 1 is the flow chart of real-time fluorescence quantitative PCR detection method；
Fig. 2 is the regular-PCR gel electrophoresis figure of the different sample lactobacillus acetotoleranses used；
Fig. 3 is the amplification curve diagram of standard items DNA quantitative fluorescent PCR；
Fig. 4 is the solubility curve figure of standard items DNA quantitative fluorescent PCR；
Fig. 5 is the canonical plotting of standard items DNA quantitative fluorescent PCR；
Fig. 6 is the sensitivity testing result of detection method；
Fig. 7 is the repeated testing result of detection method；
Fig. 8 is the accuracy testing result of detection method.
Embodiment
The present invention is described in further detail below by specific embodiment and with reference to accompanying drawing.
Embodiment 1：The detection of putrefactive microorganisms in yellow rice wine
Step 1, primer synthesizes：
The sequence of primer is：
Forward primer P1-F：5 '-TCCAGCTTATGCGGAAGCAC-3 ' (sequence is as shown in SEQ ID NO.1)；
Reverse primer P1-R：5 '-AGTCCCAGAAAGCTTACGCA-3 ' (sequence is as shown in SEQ ID NO.2)；
The primer is dispensed with pipe, and the date of packing and the Primer are indicated on pipe, 5 are carried out with sterilized water
Times dilute, as primer stoste, be put in -20 DEG C of refrigerators and preserve, directly take during use；
The primer relevant information of table 1
Step 2, the specificity of primer is identified in regular-PCR, electrophoresis and sequencing：
The nutrient solution of lactobacillus acetotolerans filtered out in becoming sour yellow rice wine sample from difference is selected, is tried using DNA of bacteria Rapid extraction
Agent box, DNA is extracted, regular-PCR is carried out as DNA profiling；
Step 2-1, regular-PCR 25ul reaction system：
The reaction system configured is put in the amplification of PCR instrument progress genetic fragment；
Step 2-2, pcr amplification reaction：
94 DEG C of pre-degeneration 4min；94 DEG C of denaturation 10s, 60 DEG C of annealing 30s, expand 40 circulations；72 DEG C of extension 10min；Take
7ulPCR products are detected with 1% agarose gel electrophoresis；
Such as Fig. 2, the lactobacillus acetotolerans DNA of different samples electrophoresis is can be seen that from the PCR electrophoretograms of lactobacillus acetotolerans
Band is single, no dimer and band becomes clear, and shows：Amplification of the primer of design for the purpose fragment in sample is respectively provided with very
Good specificity, while amplified fragments size is coincide with expected expanding fragment length, is tentatively shown：The primer specificity of design
Preferably, it is 60 DEG C to recommend annealing temperature.
Step 2-3, PCR primer sequencing.
The sequencing sequence comparing result of table 2
| Sample number into spectrum | NCBI comparison results | Similarity | GeneBank accession number |
| 12 | Lactobacillus acetotolerans | 100% | AP014808.1 |
| 13 | Lactobacillus acetotolerans | 100% | AP014808.1 |
| 14 | Lactobacillus acetotolerans | 100% | AP014808.1 |
| 15 | Lactobacillus acetotolerans | 100% | AP014808.1 |
Step 3：Standard items DNA preparation：
The bacteria suspension of the lactobacillus acetotolerans of the growth selection stage of stable development, using DNA of bacteria Rapid extraction kit, DNA is extracted,
After determining DNA concentration, the sample DNA of extraction is subjected to different multiples dilution, quantitative fluorescent PCR is carried out as DNA profiling,
Simultaneously by the bacteria suspension of lactobacillus acetotolerans, 10 times of gradient dilutions are carried out, do 6 dilution factors altogether：10-1、10-2、10-3、10-4、
10-5、10-6, select dilution factor 10-4、10-5、10-6Flat board coating is carried out, after cultivating one week, the bacterium colony for carrying out microorganism counts；
Bacterium colony count results：1.39×109CFU/mL。
Step 4, the foundation of real-time fluorescence quantitative PCR reaction system and response procedures；
The dense template DNA of known bacterium is subjected to gradient dilution, chooses 100、10-1、10-2、10-3、10-4、10-5、10-6、10-7
Totally 8 concentration gradients as template carry out quantitative fluorescent PCR, each template do 3 it is parallel；
Step 4-1, quantitative fluorescent PCR 20ul reaction system：
Step 4-2, quantitative fluorescent PCR response procedures：
95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 10s, 60 DEG C of 30s, collect fluorescence signal, expand 40 circulations；65℃—95℃
Heating, fluorescence signal is collected, draw melting curve；
Step 5, the drafting of standard curve：
The standard items DNA that will be prepared in step 3,10 times of gradient dilutions are carried out, do 8 dilution factors altogether：100、10-1、
10-2、10-3、10-4、10-5、10-6、10-7, using this 8 dilution factors as standard items DNA profiling, configure quantitative PCR body
It is and is expanded, each template 3 is parallel, is mapped with microorganism concn and corresponding threshold cycle number Ct, line of going forward side by side
Fitting, obtains standard curve；
Amplification curve, melting curve and standard curve are respectively as shown in Fig. 3, Fig. 4 and Fig. 5：There it can be seen that amplification is bent
Line, melting curve are all fine, while the R of standard curve (y=-3.5453x+43.13)2For 0.9988, amplification efficiency 91.5%,
Show that the fluorescence quantification PCR primer specificity of design is good.
Step 6, the assessment of real-time fluorescence quantitative PCR detection method.
Step 6-1, the sensitivity evaluation of real-time fluorescence quantitative PCR detection method：
The dense template DNA of known bacterium is subjected to gradient dilution, chooses 100、10-1、10-2、10-3、10-4、10-5、10-6、10-7
Totally 8 concentration gradients carry out quantitative fluorescent PCR, determine that the minimum bacterium that quantitative fluorescent PCR can be detected out is dense by reaction,
That is Monitoring lower-cut, the sensitivity of this method is evaluated；
Experimental result as shown in fig. 6, bacterium it is dense be 1.0 × 10^3 template obvious fluorescent value can also be detected by this method, follow
Number of rings (Ct) is 30.78, illustrates that this method has higher sensitivity.
Step 6-2, the repeatability and estimation of stability of real-time fluorescence quantitative PCR detection method：
The standard items DNA of the dense different extension rates of known bacterium is carried out into batch interior repetition to test, each standard items DNA sets 3
It is individual parallel, calculate the coefficient of variation of Ct values；
The amplification curve repeated in batch is as shown in Figure 7, it is seen that repeatability is preferably.Repeat to test its corresponding Ct values such as table 3 in batch
Shown, as seen from the table, the average value that the value for coefficient of variation of experiment is repeated in batch is 0.28%, shows established fluorescent quantitation
Method has good stability.The coefficient of variation that experiment is repeated in batch is much smaller than 3.0%, and error is minimum, coincidence statistics rule
Rule, fully confirm the good reproducibility of the detection method.
Repeat to test in the fluorescence quantifying PCR method batch of table 3
Step 6-3, the evaluation of the accuracy of real-time fluorescence quantitative PCR detection method：
Using the DNA sample of the dense different extension rates of known bacterium as unknown sample, quantitative fluorescent PCR, experiment with computing are carried out
Quantitative result and the dense difference of actual bacterium.
Experimental result is as shown in figure 8, it can be seen that quantitative result is close with theoretical copy number twice, on the order of magnitude
There is no difference.Illustrate that the primer and corresponding quantitative approach accuracy are high, can well realize and lactobacillus acetotolerans is quantified.
Embodiment 2：Detect the method that yellow rice wine whether there is corrupt potential quality
Step 1, primer synthesizes：
Forward primer P1-F：5 '-TCCAGCTTATGCGGAAGCAC-3 ' (sequence is as shown in SEQ ID NO.1)；
Reverse primer P1-R：5 '-AGTCCCAGAAAGCTTACGCA-3 ' (sequence is as shown in SEQ ID NO.2)；
Step 2, real-time fluorescence quantitative PCR reacts；
The genome for selecting four yellow rice wine samples carries out quantitative fluorescent PCR reaction, and reaction system includes sterilized water 8.2 μ l, SYBR
The μ l of Green I 10, the μ l of forward primer 0.4, the μ l of reverse primer 0.4, the μ l of DNA profiling 1, totally 20 μ l systems, response procedures are：
95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 10s, 60 DEG C of 30s, collect fluorescence signal, expand 40 circulations；65 DEG C -95 DEG C rise
Temperature, fluorescence signal is collected, draw melting curve.Obtained quantitative result is as shown in table 4.
The quantitative result of lactobacillus acetotolerans in the different yellow rice wine samples of table 4
| Sample number into spectrum | 1# | 2# | 3# | 4# |
| Quantitative result | 4.95E+05 | 4.76E+03 | 6.53E+02 | 31 |
Step 3, the acceleration of yellow rice wine sample is become sour；
Above yellow rice wine sample is put into quiescent culture in 30 DEG C of incubators, accelerates its process of becoming sour.After culture 50 days, to yellow rice wine
Sample carries out the extraction of genome.The genome of extraction is put in -20 DEG C of refrigerators and preserved, standby.
Step 4, real-time fluorescence quantitative PCR reacts；
Four standby genomes of yellow rice wine sample carry out quantitative fluorescent PCR reaction more than, and reaction system includes the μ l of sterilized water 8.2,
The μ l of SYBR Green I 10, the μ l of forward primer 0.4, the μ l of reverse primer 0.4, the μ l of DNA profiling 1, totally 20 μ l systems, reaction interval
Sequence is：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 10s, 60 DEG C of 30s, collect fluorescence signal, expand 40 circulations；65℃—95℃
Heating, fluorescence signal is collected, draw melting curve.Obtained quantitative result is as shown in table 5.
The quantitative result of lactobacillus acetotolerans in the different yellow rice wine samples of table 5
| Sample number into spectrum | 1# | 2# | 3# | 4# |
| Quantitative result | 1.50E+08 | 2.11E+07 | 3.08E+07 | 62 |
Quantitative result shows that, when the lactobacillus acetotolerans content in yellow rice wine is more than 1.00E+02CFU/mL, yellow rice wine is possible to out
Existing corruption, is provided with the potential that yellow rice wine becomes sour.Inventor has found, in brewing yellow rice wine or storage process, detects resistance to
When Lactobacillus lactis content is more than 1.00E+02CFU/mL, take appropriate measures, for example sterilize, make lactobacillus acetotolerans content small
In 100CFU/mL, then proceed to store or continue to store after the yellow rice wine after processing is mixed with other yellow rice wine up to standard, will not
Become sour phenomenon.
Further, since containing substantial amounts of microorganism in the yellow rice wine that becomes sour, inventor has also filtered out face in addition to lactobacillus acetotolerans
Bag lactobacillus (Lactobacillus Panis) etc..For lactobacillus panis, inventor have also been made corresponding quantitative fluorescent PCR inspection
Survey and analyze the research of yellow rice wine.Quantitative result shows that during yellow rice wine stores, the quantity of lactobacillus panis does not become substantially
Change, but the acidity of yellow rice wine but has different degrees of rise.Illustrate, detection lactobacillus panis can not directly indicate that yellow rice wine is
It is no the possibility become sour to be present.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any person skilled in the art,
Without departing from the spirit and scope of the present invention, various change and modification can all be done, therefore protection scope of the present invention should be with
What claims were defined is defined.
Claims (10)
- A kind of 1. method for controlling yellow rice wine quality, it is characterised in that methods described is to detect and control the lactobacillus acetotolerans in yellow rice wine Content.
- 2. according to the method for claim 1, it is characterised in that methods described is containing for the lactobacillus acetotolerans in control yellow rice wine Amount is less than 100CFU/mL.
- 3. according to the method for claim 1, it is characterised in that acidproof using fluorescence quantitative PCR detection during the detection Lactobacillus.
- 4. according to the method for claim 1, it is characterised in that methods described is to work as the lactobacillus acetotolerans detected in yellow rice wine Content more than 1.00E+02CFU/mL when, take measures to reduce the content of lactobacillus acetotolerans to being less than 100CFU/mL, so Continue the yellow rice wine after storage processing afterwards or continue to store after the yellow rice wine after processing is mixed with other yellow rice wine up to standard.
- A kind of 5. method of quick detection yellow rice wine putrefactive microorganisms, it is characterised in that methods described is to use quantitative fluorescent PCR Detect lactobacillus acetotolerans.
- 6. according to the method for claim 5, it is characterised in that the specificity used during the fluorescence quantitative PCR detection Primer pair is any pair in primer pair P1, P2, P3, P4 or P5.
- A kind of 7. method for detecting yellow rice wine, it is characterised in that methods described is to detect the content of the lactobacillus acetotolerans in yellow rice wine, is led to The presence situation analysis yellow rice wine for crossing lactobacillus acetotolerans whether there is the danger become sour.
- 8. according to the method for claim 7, it is characterised in that methods described uses the resistance to yogurt of fluorescence quantitative PCR detection The presence or absence of bacillus, or the content of lactobacillus acetotolerans.
- 9. according to the method for claim 7, it is characterised in that methods described is the content of quantitative detection lactobacillus acetotolerans, When lactobacillus acetotolerans is more than 1.00E+02CFU/mL, take measures to reduce the content of lactobacillus acetotolerans.
- 10. a species-specific primer pair, the specific primer is to being any in primer pair P1, P2, P3, P4 or P5 A pair.
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| CN109192247A (en) * | 2018-09-28 | 2019-01-11 | 山东衡昊信息技术有限公司 | A kind of yellow rice wine sterilising microbe content curve generation method |
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Application publication date: 20180130 |
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