CN117511931A - Method for extracting total DNA of Daqu at high product temperature in white spirit fermentation process - Google Patents
Method for extracting total DNA of Daqu at high product temperature in white spirit fermentation process Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 51
- 230000004151 fermentation Effects 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 33
- 238000000227 grinding Methods 0.000 claims abstract description 18
- 238000005406 washing Methods 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims description 12
- 102000016943 Muramidase Human genes 0.000 claims description 11
- 108010014251 Muramidase Proteins 0.000 claims description 11
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 11
- 241001052560 Thallis Species 0.000 claims description 11
- 235000010335 lysozyme Nutrition 0.000 claims description 11
- 239000000047 product Substances 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000006228 supernatant Substances 0.000 claims description 10
- 239000011324 bead Substances 0.000 claims description 9
- 239000011521 glass Substances 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 108010067770 Endopeptidase K Proteins 0.000 claims description 6
- 238000011068 loading method Methods 0.000 claims description 6
- 229960000274 lysozyme Drugs 0.000 claims description 6
- 239000004325 lysozyme Substances 0.000 claims description 6
- 229940051921 muramidase Drugs 0.000 claims description 5
- 239000002689 soil Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 235000020097 white wine Nutrition 0.000 claims description 2
- 238000000605 extraction Methods 0.000 abstract description 19
- 244000005700 microbiome Species 0.000 abstract description 14
- 238000012163 sequencing technique Methods 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 4
- 238000001556 precipitation Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 52
- 238000007400 DNA extraction Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 239000000796 flavoring agent Substances 0.000 description 3
- 235000019634 flavors Nutrition 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 108091093088 Amplicon Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000235342 Saccharomycetes Species 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012268 genome sequencing Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000013124 brewing process Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
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- 150000002148 esters Chemical class 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 235000014101 wine Nutrition 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a method for extracting total DNA of Daqu at high temperature in a white spirit fermentation process, and belongs to the technical field of bioengineering. The invention researches a high-efficiency extraction method of high-temperature Daqu total DNA in a fermentation process, and the total DNA of the high-temperature Daqu microorganisms in the fermentation process is obtained by combining an innovative DNA pretreatment extraction method with a low-temperature precooling grinding method and finally performing precipitation, washing and collection. The high-efficiency extraction method of total DNA provided by the invention can be applied to a high-temperature Daqu sample in the fermentation process, and the total DNA with high concentration and high purity can be separated from the high-temperature Daqu in the fermentation process, so that the method can be directly used for metagenome analysis, and can effectively solve the problem that the existing extraction method of the DNA of the white spirit Daqu cannot meet the requirement of metagenome sequencing of the high-temperature Daqu in the fermentation process.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for extracting total DNA of high-temperature Daqu in a white spirit fermentation process and application thereof.
Background
Daqu is a starter for brewing white spirit, provides saccharification force and liquefaction force for the white spirit brewing process, and is crucial to the formation of flavor in white spirit, so that the quality of Daqu directly determines the yield and quality of white spirit. The microorganisms in the Daqu mainly comprise bacteria, mould and saccharomycetes, wherein the bacteria are the main power for producing aroma, the mould mainly plays a role in saccharification, and the saccharomycetes have the capacity of alcohol and ester production. It can be seen that functional microorganisms in Daqu play an important role in brewing white spirit.
However, only 1% of microorganisms in the environment can be cultured under the experimental condition, and the limitation results in that information of very few microorganisms in the environment can be obtained, so that the ecological distribution situation of the microorganisms in the wine cheese environment can not be fully reflected, and along with the wide application of culture-free technology (DNA sequence homology analysis, PCR-DGGE, clone library, second generation sequencing and the like), the knowledge of the diversity and structure of the finished white spirit Daqu microorganism community is relatively clear. At present, research on Daqu microorganisms in a fermentation process is mainly focused on an amplicon sequencing method, no report on macro genome analysis of high-temperature Daqu in the fermentation process of Daqu is seen, and a macro genome sequencing technology can be used for deeply knowing a gene function and can more accurately reflect active microorganisms actually contributing to white spirit Daqu.
Research shows that the existing reported method for extracting Daqu DNA can not meet the requirement of macro genome sequencing of high-temperature Daqu in the fermentation process, for example, patent CN113215146A can extract the total DNA of finished Daqu for metagenome analysis, but the method can not extract the total DNA of high-temperature Daqu in the fermentation process; the paper "improved method for extracting total DNA of sesame-flavored white spirit high-temperature Daqu microorganisms [ J ]. Brewing, 2012,39 (04): 33-37" extracted DNA can be used for amplicon sequencing, and is extracted fermented high-temperature sesame-flavored Daqu, but the DNA of the high-temperature Daqu in the process of extracting and fermenting according to the method can not meet the condition of metagenome sequencing.
Therefore, a method for efficiently extracting total DNA of Daqu in the fermentation process is urgently needed, and the method is used for metagenomic analysis of high-temperature Daqu microorganisms in the fermentation process, and has important significance for researching the generation mechanism of important flavor substances of white spirit high-temperature Daqu in the fermentation process and improving the quality of Daqu.
Disclosure of Invention
The technical problem to be solved by the invention is that the existing method for extracting the DNA of the Daqu of white spirit can not meet the requirement of sequencing the macro genome of the Daqu at high temperature in the fermentation process.
The technical scheme adopted for solving the technical problems is as follows: the method for extracting the total DNA of the Daqu at high product temperature in the white spirit fermentation process comprises the following steps:
a. taking high-temperature Daqu during the fermentation process of white spirit, rapidly crushing, and freezing and preserving at-80 ℃ for later use;
b. mixing 50-100g of Daqu prepared in the step a with sterile PBS buffer solution uniformly, loading into a sterile centrifuge tube for centrifugation, and taking supernatant;
c. c, loading the supernatant obtained in the step b into a sterile centrifuge tube for centrifugation, and removing the supernatant to obtain thalli;
d. uniformly mixing 1mL of PBS buffer solution with thalli, loading into a sterile centrifuge tube, adding 10 mu L of muramidase, lysozyme and proteinase K, and finally adding glass beads for grinding;
e. and (5) removing protein of the ground thalli by adopting a FastDNA Spin Kit for Soil kit, and washing to obtain the total DNA of the high-temperature Daqu in the white wine fermentation process.
In the step a, the crushed Daqu is sieved by a 20-mesh sieve.
In the step b, the mass ratio of the sterile PBS buffer to the yeast prepared in the step a is 1:8-10.
In the step b, the rotational speed of centrifugation is 1500-1800rpm, and the centrifugation time is 5min.
In the step c, the rotational speed of the centrifugation was 12000rpm, and the centrifugation time was 5 minutes.
In the step d, the addition amount of the muramidase is 20g/L, the addition amount of the lysozyme is 50g/L, and the addition amount of the proteinase K is 10g/L.
In the step d, the glass beads are sterile glass beads with the diameter of 0.2-1mm, and the adding amount is 0.2-0.5g.
In the step d, the sterile centrifuge tube is subjected to grinding for 10s by a grinding instrument, then refrigerated at-20 ℃ for 2min and then is subjected to grinding for 10s.
The beneficial effects of the invention are as follows: the invention researches a high-efficiency extraction method of high-temperature Daqu total DNA in a fermentation process, and the total DNA of the high-temperature Daqu microorganisms in the fermentation process is obtained by combining an innovative DNA pretreatment extraction method with a low-temperature precooling grinding method and finally performing precipitation, washing and collection. The extraction method is convenient, quick and efficient to operate; the extraction process is simple and easy to operate, and the defect that the existing DNA extraction method can not extract the total DNA of the Daqu at high product temperature in the fermentation process is overcome; the operation is rapid, and the extraction of the total DNA of the Daqu can be completed within 2 hours; the concentration of the extracted distilled spirit Daqu is as high as 30-100ng/L, the purity is high, and the A260/A280 value is 1.8-2.0.
The high-efficiency extraction method of total DNA provided by the invention can be applied to a high-temperature Daqu sample in the fermentation process, and solves the problems that the microorganism concentration of the high-temperature Daqu in the fermentation process is low and the total DNA is difficult to extract. The method provided by the invention can be used for separating the total DNA with high concentration and high purity from the high-temperature Daqu in the fermentation process for the first time, and can be directly used for metagenomic analysis. The invention can provide technical support for the macro-genome information analysis of microorganisms in the high-temperature Daqu in the fermentation process, and has important significance for researching the generation mechanism of important flavor substances in the fermentation process of Daqu and improving the quality of Daqu.
Drawings
FIG. 1 is a bar graph showing the total DNA concentration extracted in examples 1-2 and comparative examples 1-2 according to the present invention.
Detailed Description
The technical scheme of the invention can be implemented in the following way.
The method for extracting the total DNA of the high-temperature Daqu in the white spirit fermentation process comprises the following specific steps:
(1) Taking a high-temperature Daqu sample in the white spirit fermentation process, rapidly crushing, and freezing and preserving at-80 ℃ for later use;
(2) Weighing crushed Daqu, adding into a triangular flask containing sterile PBS buffer solution, and shaking for 30min;
(3) Transferring the solution to a 50mL sterile centrifuge tube, and centrifuging at 1500-1800rpm for 5min;
(4) Transferring the supernatant to a new sterile centrifuge tube, centrifuging at 12000rpm for 5min, and discarding the supernatant until all PBS Daqu solution is collected to obtain thalli;
(5) 1mL of PBS buffer was added to the cells, the cells were lysed and transferred to a 2mL sterile tube, 10. Mu.L of lywallase (20 g/L), lysozyme (50 g/L) and proteinase K (10 g/L) were added, and 0.2g of glass beads were added;
(6) Grinding a 2mL centrifuge tube in a grinder for 10s, refrigerating for 2min at-20 ℃, continuously grinding for 10s, and repeatedly grinding once;
(7) And then deproteinizing by combining with a FastDNA Spin Kit for Soil kit, and washing to obtain the total DNA.
Wherein, the step (1) is to crush the yeast with the thickness of 20 meshes;
wherein, the mass of the Daqu is 50-100g in the step (2);
wherein, in the step (2), PBS buffer solution is added and stirred, and the mass ratio of the PBS buffer solution to the Daqu is 1:8-10;
wherein, the thallus dissolving in the step (5) needs to be fully blown and evenly mixed by a sterile suction head, and the added glass beads are sterile and have the diameter of 0.2-1mm;
wherein, the grinding time in the step (6) is 10s, so that the DNA is prevented from generating high temperature due to long-time oscillation;
wherein, the step (6) is carried out once grinding and then is refrigerated for 2min at the temperature of minus 20 ℃ to reduce the temperature of the centrifuge tube and prevent continuous oscillation from generating high temperature so that DNA is broken into small fragments;
wherein, all the operation steps use aseptic materials;
the invention also provides the application of the method for rapidly extracting the total DNA of the high-temperature Daqu in the fermentation process of the white spirit in the extraction of the total DNA of the high-temperature Daqu in the fermentation process.
The technical scheme and effect of the present invention will be further described by practical examples.
Examples
EXAMPLE 1 Total DNA extraction of wuliangye Packed yeast at 60℃for 10 days of fermentation
1. Sampling
From 3 wuliangye bags Qu Qufang fermented for 10 days, 1 wuliangye bag Qu Yangpin with temperature of about 60 ℃ is randomly extracted from each yeast room, and is put into a sterile bag, crushed to the size of 20 meshes by a crusher, put into the sterile bag, and rapidly put into a refrigerator with the temperature of-80 ℃ for standby.
2. Extraction of Total RNA
The extraction steps are as follows:
(1) Weighing 50g of crushed high-temperature Daqu during fermentation, adding into a triangular flask containing sterile PBS buffer solution, and shaking for 30min;
(2) Transferring the solution to a 50mL sterile centrifuge tube, and centrifuging at 1800rpm for 5min;
(3) Transferring the supernatant to a new sterile centrifuge tube, centrifuging at 12000rpm for 5min, and discarding the supernatant to obtain thalli;
(4) Repeating the steps (2) and (3) for 6 times, and collecting all thalli;
(5) Adding 1mLPBS buffer solution into the thalli, blowing with a sterile suction head to enable the thalli to be fully dissolved in the buffer solution, transferring to a 2mL sterile tube, adding 10 mu L of muramidase, lysozyme and proteinase K, and adding glass beads with the diameter of 0.2-1mm;
(6) Grinding a 2mL centrifuge tube in a grinder for 10s, refrigerating for 2min at-20 ℃, continuously grinding for 10s, and repeatedly grinding once;
(7) And then deproteinizing by combining with the FastDNA Spin Kit for Soil kit, and adding 60 mu L of eluent for washing to obtain the total DNA.
3. Detection of Total DNA
The total DNA extracted was extracted by 1. Mu.L, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scitic company, and the results are shown in Table 1 and FIG. 1, and it was found that the wuliangye bag Bao Quzong DNA concentration at 60℃for 10 days of fermentation was 30-100 ng/. Mu.L, and the A260/A280 value was 1.8-2.0, indicating that both the DNA purity and the DNA concentration were high.
EXAMPLE 2 Total DNA extraction of wuliangye Packed in yeast at 59℃for 15 days of fermentation
1. Sampling
From 3 wuliangye bags Qu Qufang fermented for 15 days, 1 wuliangye bag Qu Yangpin with temperature of about 59 ℃ is randomly extracted from each yeast room, and is put into a sterile bag, crushed to the size of 20 meshes by a crusher, put into the sterile bag, and rapidly put into a refrigerator with the temperature of minus 80 ℃ for standby.
2. The extraction of total DNA was carried out in the same manner as in example 1.
3. Detection of Total DNA
The total DNA extracted was extracted by 1. Mu.L, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scitic company, and the results are shown in Table 1 and FIG. 1, and it was found that the wuliangye bag Bao Quzong DNA concentration at 59℃for 15 days of fermentation was 30-100 ng/. Mu.L, and the A260/A280 value was 1.8-2.0, indicating that both the DNA purity and the DNA concentration were high.
Comparative example 1
1. Sampling was performed as in example 1.
2. Extraction of total DNA the extraction step was performed according to the daqu DNA extraction step described in patent CN113215146 a.
3. Detection of Total DNA
The total DNA extracted by 1. Mu.L was extracted, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scitic company, and the results are shown in Table 1 and FIG. 1, and it was found that the wuliangye bag Bao Quzong DNA concentration at 60℃for 10 days of fermentation extracted by this method was less than 10 ng/. Mu.L, and the A260/A280 value was not between 1.8 and 2.0, indicating that the DNA concentration was low and purity was not high, and that the requirement for metagenomic sequencing could not be satisfied.
Comparative example 2
1. Sampling was performed as in example 2.
2. Extraction of total DNA the extraction procedure was pre-treated according to the daqu DNA extraction procedure described in patent CN113215146a, combined with FastDNA Spin Kit for Soil kit for DNA extraction.
3. Detection of Total DNA
The total DNA extracted by 1. Mu.L was extracted, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scitic company, and the results are shown in Table 1 and FIG. 1, and it was found that the wuliangye bag Bao Quzong DNA concentration at 59℃for 15 days of fermentation extracted by this method was less than 10 ng/. Mu.L, and the A260/A280 value was not between 1.8 and 2.0, indicating that the DNA concentration was low and purity was not high, and that the requirement for metagenomic sequencing could not be satisfied.
TABLE 1 results of DNA concentration and purity measurements for examples and comparative examples packages Bao Quzong
The method for extracting the total DNA of the high-temperature Daqu in the fermentation process is not only suitable for the aroma type wuliangye bag Bao Qu, but also suitable for the extraction of the total DNA of the high-temperature Daqu in various fermentation processes and finished white spirit Daqu.
Claims (8)
1. The method for extracting the total DNA of the Daqu at high product temperature in the white spirit fermentation process is characterized by comprising the following steps of:
a. taking high-temperature Daqu during the fermentation process of white spirit, rapidly crushing, and freezing and preserving at-80 ℃ for later use;
b. mixing 50-100g of Daqu prepared in the step a with sterile PBS buffer solution uniformly, loading into a sterile centrifuge tube for centrifugation, and taking supernatant;
c. c, loading the supernatant obtained in the step b into a sterile centrifuge tube for centrifugation, and removing the supernatant to obtain thalli;
d. uniformly mixing 1mL of PBS buffer solution with thalli, loading into a sterile centrifuge tube, adding 10 mu L of muramidase, lysozyme and proteinase K, and finally adding glass beads for grinding;
e. and (5) removing protein of the ground thalli by adopting a FastDNA Spin Kit for Soil kit, and washing to obtain the total DNA of the high-temperature Daqu in the white wine fermentation process.
2. The method for extracting total DNA of Daqu liquor fermentation process high temperature product according to claim 1, which is characterized in that: in the step a, the crushed Daqu is sieved by a 20-mesh sieve.
3. The method for extracting total DNA of Daqu liquor fermentation process high temperature product according to claim 1, which is characterized in that: in step b, the mass ratio of the sterile PBS buffer to the yeast for standby in step a is 1:8-10.
4. The method for extracting total DNA of Daqu liquor fermentation process high temperature product according to claim 1, which is characterized in that: in step b, the rotational speed of centrifugation is 1500-1800rpm, and the centrifugation time is 5min.
5. The method for extracting total DNA of Daqu liquor fermentation process high temperature product according to claim 1, which is characterized in that: in step c, the rotational speed of the centrifugation was 12000rpm, and the centrifugation time was 5min.
6. The method for extracting total DNA of Daqu liquor fermentation process high temperature product according to claim 1, which is characterized in that: in step d, the addition amount of the muramidase is 20g/L, the addition amount of the lysozyme is 50g/L, and the addition amount of the proteinase K is 10g/L.
7. The method for extracting total DNA of Daqu liquor fermentation process high temperature product according to claim 1, which is characterized in that: in the step d, the glass beads are sterile glass beads with the diameter of 0.2-1mm, and the adding amount is 0.2-0.5g.
8. The method for extracting total DNA of Daqu liquor fermentation process high temperature product according to claim 1, which is characterized in that: in the step d, the sterile centrifuge tube is subjected to grinding for 10s by a grinding instrument, then is refrigerated at-20 ℃ for 2min, and then is subjected to grinding for 10s.
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