CN117511931A - Method for extracting total DNA of Daqu at high product temperature in white spirit fermentation process - Google Patents

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

Extraction method of total DNA from high-grade Wen Daqu during liquor fermentation process

Technical field

The invention belongs to the field of bioengineering technology, and specifically relates to a method for extracting total DNA of high-grade warm Daqu during liquor fermentation and its application.

Background technique

Daqu is a fermentation agent for liquor brewing. It provides saccharification and liquefaction power for the liquor brewing process. It is also crucial to the formation of flavor in liquor. Therefore, the quality of Daqu directly determines the yield and quality of liquor. The microorganisms in Daqu mainly include bacteria, molds and yeasts. Bacteria are the main driving force for aroma production, molds mainly play the role of saccharification, and yeasts have the ability to wine and produce esters. It can be seen that the functional microorganisms in Daqu play an important role in liquor brewing.

However, only 1% of the microorganisms in the environment can be cultured under experimental conditions. This limitation results in that only a very small number of microorganisms in the environment can be obtained. Therefore, it cannot fully reflect the ecological distribution of microorganisms in the wine and cheese environment. With the introduction of culture-free With the widespread application of technology (DNA sequence homology analysis, PCR-DGGE, clone library, second-generation sequencing, etc.), the understanding of the diversity and structure of the microbial community of Daqu in finished liquor has been relatively clear. Current research on Daqu microorganisms during the fermentation process mainly focuses on amplicon sequencing methods. There are no reports on metagenomic analysis of high-quality Daqu during the fermentation process. Metagenomic sequencing technology can provide an in-depth understanding of gene functions and more accurately reflect the true content of Daqu in liquor. Contributing active microorganisms.

The study found that the existing reported Daqu DNA extraction methods cannot meet the metagenomic sequencing needs of Daqu with high product temperature during fermentation. For example, the patent CN113215146A can extract the total DNA of finished Daqu for metagenomic analysis. However, this method cannot extract the total DNA of Daqu with high product temperature during fermentation; the paper " Improved method for extracting total microbial DNA from Wen Daqu, a high-grade sesame-flavored liquor [J]. Brewing, 2012, 39(04): 33-37 "The extracted DNA can be used for amplicon sequencing, and is extracted from high-temperature sesame seeds that have been fermented. Flavor Daqu, but according to this method, the DNA extracted from high-grade warm Daqu during the fermentation process cannot meet the metagenomic sequencing conditions.

Therefore, there is an urgent need for an efficient method for extracting the total DNA of Daqu during the fermentation process, which can be used for metagenomic analysis of microorganisms in high-quality Daqu during the fermentation process. It is of great significance for the study of the production mechanism of important flavor substances of high-quality and warm Daqu during the fermentation process and for improving the quality of Daqu. .

Contents of the invention

The technical problem to be solved by the present invention is that the existing DNA extraction method of liquor Daqu cannot meet the metagenomic sequencing requirements of high-grade and high-temperature Daqu during the fermentation process.

The technical solution adopted by the present invention to solve the technical problem is: a method for extracting the total DNA of high-grade warm Daqu during the liquor fermentation process, including the following steps:

a. Take Wen Daqu, a high-quality product from the liquor fermentation process, crush it quickly and freeze it at -80°C for later use;

b. Mix 50-100g of Daqu reserved in step a with sterile PBS buffer, put it into a sterile centrifuge tube, and centrifuge, and take the supernatant;

c. Put the supernatant obtained in step b into a sterile centrifuge tube and centrifuge, remove the supernatant to obtain bacterial cells;

d. Mix 1 mL of PBS buffer and bacteria and put them into a sterile centrifuge tube, then add 10 μL of wall-lysing enzyme, lysozyme and proteinase K, and finally add glass beads for grinding;

e. Use the FastDNA Spin Kit for Soil kit to remove the protein from the ground bacteria and wash it to obtain the total DNA of high-grade Wen Daqu during the liquor fermentation process.

In the above step a, the size of the crushed Daqu should be passed through a 20-mesh sieve.

In the above step b, the mass ratio of sterile PBS buffer to the Daqu prepared in step a is 1:8-10.

In the above step b, the centrifugation speed is 1500-1800 rpm, and the centrifugation time is 5 minutes.

In the above step c, the centrifugation speed is 12000 rpm and the centrifugation time is 5 minutes.

In the above step d, the addition amount of wall-lytic enzyme is 20g/L, the addition amount of lysozyme is 50g/L, and the addition amount of proteinase K is 10g/L.

In the above step d, the glass beads are sterile glass beads with a diameter of 0.2-1mm, and the added amount is 0.2-0.5g.

In step d above, the grinding method is to grind the sterile centrifuge tube in a grinder for 10 seconds, then refrigerate it at -20°C for 2 minutes, and then continue grinding for 10 seconds.

The beneficial effects of the present invention are: the present invention studies an efficient extraction method of total DNA from Daqu with a high product temperature during the fermentation process. Through an innovative DNA pre-treatment extraction method combined with a low-temperature pre-cooling grinding method, and finally collection through precipitation and washing, Daqu with a high product temperature during the fermentation process is obtained. Total DNA of microorganisms. The extraction method of the present invention is easy to operate, fast and efficient; the extraction process is simple and easy to operate, and overcomes the shortcoming of the existing DNA extraction method that cannot extract the total DNA of Daqu with high product temperature during the fermentation process; and the operation is fast, and the total DNA of Daqu can be completed within 2 hours. Extraction; the concentration of extracted liquor Daqu is as high as 30-100ng/L, and the purity is high, with an A260/A280 value of 1.8-2.0.

The efficient extraction method of total DNA provided by the invention can be applied to Daqu samples with high product temperature during the fermentation process, and solves the problem that the concentration of microorganisms in Daqu with high product temperature during the fermentation process is low and the total DNA is difficult to extract. This invention separates for the first time high-concentration and high-purity total DNA from high-quality Daqu during the fermentation process, which can be directly used for metagenomic analysis. The present invention can provide technical support for the metagenomic information analysis of microorganisms in high-grade temperature Daqu during the fermentation process, and is of great significance for the research on the production mechanism of important flavor substances during the Daqu fermentation process and for improving the quality of Daqu.

Description of drawings

Figure 1 is a histogram of the total DNA concentration extracted in Example 1-2 and Comparative Example 1-2 of the present invention.

Detailed ways

The technical solution of the present invention can be implemented specifically in the following manner.

The specific steps for extracting total DNA from high-grade Wen Daqu during the liquor fermentation process are as follows:

(1) Take a sample of high-grade Wen Daqu from the liquor fermentation process, crush it quickly, and freeze it at -80°C for later use;

(2) Weigh the crushed Daqu, add it to an Erlenmeyer flask containing sterile PBS buffer, and shake frequently for 30 minutes;

(3) Transfer the solution to a 50mL sterile centrifuge tube and centrifuge at 1500-1800rpm for 5 minutes;

(4) Transfer the supernatant to a new sterile centrifuge tube, centrifuge at 12,000 rpm for 5 minutes, and discard the supernatant until all the PBS Daqu solution is collected to obtain bacterial cells;

(5) Add 1mL of PBS buffer to the bacterial cells, dissolve the bacterial cells and transfer to a 2mL sterile tube, then add 10μl of wall-lysing enzyme (20g/L), lysozyme (50g/L) and proteinase K ( 10g/L), and add 0.2g glass beads;

(6) Grind the 2mL centrifuge tube in a grinder for 10 seconds, refrigerate at -20°C for 2 minutes, continue grinding for 10 seconds, and repeat grinding once;

(7) Subsequently combine the FastDNA Spin Kit for Soil kit to remove protein and wash to obtain the total DNA.

Wherein, the above-mentioned step (1) crushes Daqu to a thickness of passing through a 20-mesh sieve;

Among them, the above-mentioned step (2) weighs the mass of Daqu to be 50-100g;

Wherein, in the above step (2), PBS buffer is added and stirred, and the mass ratio of PBS buffer to Daqu is 1:8-10;

Among them, in the above step (5), dissolving the bacterial cells requires full pipetting and mixing with a sterile pipette, and the glass beads added are sterile and have a diameter of 0.2-1mm;

Among them, the grinding time in step (6) above is 10 seconds to prevent DNA from oscillating for a long time and causing high temperature;

Among them, after grinding once in the above step (6), it needs to be refrigerated at -20°C for 2 minutes to lower the temperature of the centrifuge tube and prevent the DNA from being broken into small fragments due to the high temperature generated by continued shaking;

Among them, all the above operation steps use sterile materials;

The present invention also provides the use of the above method for rapid extraction of total DNA from high-grade and high-grade temperature Daqu during the fermentation process of liquor in the extraction of total DNA from high-grade and high-grade temperature Daqu during the fermentation process.

The technical solutions and effects of the present invention will be further described below through practical examples.

Example

Example 1 Extraction of total DNA from Wuliangye Baobaoqu fermented for 10 days at a temperature of 60°C

1. Sampling

From the three Wuliangye Baoqu rooms that were fermented for 10 days, randomly select a sample of Wuliangye Baoqu with a temperature of about 60°C from each room, put it into a sterile bag, and crush it with a grinder until it passes 20 mesh. Sieve size, put it in a sterile bag, and quickly place it in a -80°C refrigerator for later use.

2. Extraction of total RNA

The extraction steps are:

(1) Weigh 50g of crushed fermentation process high-grade Wen Daqu, add it to an Erlenmeyer flask containing sterile PBS buffer, and shake frequently for 30 minutes;

(2) Transfer the above solution to a 50mL sterile centrifuge tube and centrifuge at 1800rpm for 5 minutes;

(3) Transfer the supernatant to a new sterile centrifuge tube, centrifuge at 12,000 rpm for 5 minutes, discard the supernatant, and obtain bacterial cells;

(4) Repeat the above steps (2) and (3) for a total of 6 times to collect all the bacteria;

(5) Add 1 mL of PBS buffer to the above bacterial cells, pipet with a sterile tip to fully dissolve the bacterial cells in the buffer, and transfer to a 2 mL sterile tube, then add 10 μL of wall-lysing enzyme, lysozyme and proteinase K , and add glass beads with a diameter of 0.2-1mm;

(6) Grind the 2mL centrifuge tube in a grinder for 10 seconds, refrigerate at -20°C for 2 minutes, continue grinding for 10 seconds, and repeat grinding once;

(7) Subsequently combine the FastDNA Spin Kit for Soil kit to remove protein, add 60 μL of eluent for washing, and obtain the total DNA.

3. Detection of total DNA

Take 1 μL of the extracted total DNA and measure its purity and concentration using Nanodrop one from Thermo Scientific. The results are shown in Table 1 and Figure 1. It can be seen that the total DNA concentration of Wuliangye Baobaoqu fermented at 60°C for 10 days is 30-100ng. /μL, the A260/A280 value is 1.8-2.0, indicating high DNA purity and concentration.

Example 2 Extraction of total DNA from Wuliangye Baobaoqu fermented for 15 days at a temperature of 59°C

1. Sampling

From the three Wuliangye Bao Baoqu rooms fermented for 15 days, randomly select a Wuliangye Bao Baoqu sample with a temperature of about 59°C from each koji room, put it into a sterile bag, and crush it with a grinder until it passes 20 mesh. Sieve size, put it in a sterile bag, and quickly place it in a -80°C refrigerator for later use.

2. Extraction of total DNA, the specific operation is the same as in Example 1.

3. Detection of total DNA

Take 1 μL of the extracted total DNA and measure its purity and concentration using Nanodrop one from Thermo Scientific. The results are shown in Table 1 and Figure 1. It can be seen that the total DNA concentration of Wuliangye Baobaoqu fermented at 59°C for 15 days is 30-100ng. /μL, the A260/A280 value is 1.8-2.0, indicating high DNA purity and concentration.

Comparative example 1

1. Sampling is the same as in Example 1.

2. Extraction of total DNA. The extraction steps are based on the Daqu DNA extraction steps described in patent CN113215146A.

3. Detection of total DNA

Take 1 μL of the extracted total DNA and measure its purity and concentration using Nanodrop one from Thermo Scientific. The results are shown in Table 1 and Figure 1. It can be seen that the total DNA of Wuliangye Baobaoqu extracted by this method and fermented for 10 days at a temperature of 60°C The concentration is less than 10ng/μL, and the A260/A280 value is not between 1.8-2.0, indicating that the DNA concentration is low and the purity is not high, and it cannot meet the requirements of metagenomic sequencing.

Comparative example 2

1. Sampling, sampling is the same as in Example 2.

2. Extraction of total DNA, the extraction steps are pre-processed according to the Daqu DNA extraction steps described in patent CN113215146A, and DNA extraction is performed in combination with the FastDNA Spin Kit for Soil kit.

3. Detection of total DNA

Take 1 μL of the total DNA extracted and measure its purity and concentration using Nanodrop one from Thermo Scientific. The results are shown in Table 1 and Figure 1. It can be seen that the total DNA of Wuliangye Baobaoqu extracted by this method and the temperature was 59°C for 15 days. The concentration is less than 10ng/μL, and the A260/A280 value is not between 1.8-2.0, indicating that the DNA concentration is low and the purity is not high, and it cannot meet the requirements of metagenomic sequencing.

Table 1 Examples and Comparative Examples Baobaoqu total DNA concentration and purity detection results

The method for extracting total DNA from high-grade and high-grade Daqu in the fermentation process of the present invention is not only suitable for strong-flavor Wuliangye Baobao Qu, but is also suitable for extraction of high-grade and warm total DNA from various fermentation processes and finished liquor Daqu. The method of the invention extracts quickly and extracts total microorganisms. The DNA has high concentration and purity, and can effectively extract microorganisms in the sample.

Claims (8)

1. A method for extracting total DNA from high-grade Wen Daqu during the liquor fermentation process, which is characterized by including the following steps: a. Take Wen Daqu, a high-quality product from the liquor fermentation process, crush it quickly and freeze it at -80°C for later use; b. Mix 50-100g of Daqu reserved in step a with sterile PBS buffer, put it into a sterile centrifuge tube, and centrifuge, and take the supernatant; c. Put the supernatant obtained in step b into a sterile centrifuge tube and centrifuge, remove the supernatant to obtain bacterial cells; d. Mix 1 mL of PBS buffer and bacteria and put them into a sterile centrifuge tube, then add 10 μL of wall-lysing enzyme, lysozyme and proteinase K, and finally add glass beads for grinding; e. Use the FastDNA Spin Kit for Soil kit to remove the protein from the ground bacteria and wash it to obtain the total DNA of high-grade Wen Daqu during the liquor fermentation process. 2. The method for extracting the total DNA of high-grade and warm Daqu during the liquor fermentation process according to claim 1, characterized in that: in step a, the Daqu is crushed to a thickness that passes through a 20-mesh sieve. 3. The method for extracting total DNA of high-grade and high-temperature Daqu during liquor fermentation according to claim 1, characterized in that: in step b, the mass ratio of sterile PBS buffer to the Daqu prepared in step a is 1:8-10. 4. The method for extracting total DNA from high-grade warm Daqu during liquor fermentation according to claim 1, characterized in that in step b, the centrifugal speed is 1500-1800 rpm, and the centrifugation time is 5 min. 5. The method for extracting total DNA from high-grade warm Daqu during liquor fermentation according to claim 1, characterized in that in step c, the centrifugation speed is 12000 rpm and the centrifugation time is 5 min. 6. The method for extracting total DNA of high-grade warm Daqu during liquor fermentation according to claim 1, characterized in that: in step d, the addition amount of wall-lytic enzyme is 20g/L, the addition amount of lysozyme is 50g/L, and the protease amount is 50g/L. The amount of K added is 10g/L. 7. The method for extracting total DNA of high-grade warm Daqu during liquor fermentation according to claim 1, characterized in that in step d, the glass beads are sterile glass beads with a diameter of 0.2-1 mm, and the addition amount is 0.2-0.5 g. 8. The method for extracting total DNA from high-quality Daqu in the liquor fermentation process according to claim 1, characterized in that: in step d, the grinding method is to grind the sterile centrifuge tube in a grinder for 10 seconds and then refrigerate it at -20°C for 2 minutes. Then continue grinding for 10 seconds.