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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Abstract
Description
技术领域Technical field
本发明属于生物工程技术领域,具体涉及一种白酒发酵过程高品温大曲总DNA的提取方法及其应用。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.
背景技术Background technique
大曲是白酒酿造的发酵剂,为白酒酿造过程提供了糖化力与液化力,同时对白酒中风味的形成至关重要,因此大曲的品质直接决定了白酒的产量与质量。大曲中的微生物主要包括细菌、霉菌与酵母菌,其中细菌是产香的主要动力,霉菌主要起糖化作用,酵母菌具有酒化以及产酯能力。可见大曲中的功能微生物在白酒酿造中起着重要作用。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.
然而环境中仅有1%的微生物在实验条件下可培养,该局限性导致只能获得环境中极少数微生物的信息,因此不能充分反映微生物在酒酪环境中的生态分布情况,随着免培养技术(DNA序列同源分析、PCR-DGGE,克隆文库,二代测序等)的广泛应用,对成品白酒大曲微生物群落多样性和结构的认识已经相对清晰。目前对发酵过程大曲微生物的研究主要集中在扩增子测序的方法,未见大曲发酵过程高品温大曲宏基因组分析的报道,而宏基因组测序技术能够深入了解基因功能,能更准确反映白酒大曲中真正具有贡献的活性微生物。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.
研究发现,现有报道大曲DNA提取方法不能满足发酵过程高品温大曲宏基因组测序需求,如专利CN113215146A能够提取成品大曲总DNA用于宏基因组分析,然而此方法不能提取发酵过程高品温大曲总DNA;论文“芝麻香型白酒高品温大曲微生物总DNA提取的改良方法[J].酿酒,2012,39(04):33-37”提取的DNA能够用于扩增子测序,且是提取的发酵完成的高温芝麻香型大曲,但是按照其方法提取发酵过程高品温大曲的DNA不能满足宏基因组测序条件。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.
因此,急需一种高效提取发酵过程大曲总DNA的方法,将其用于发酵过程高品温大曲微生物的宏基因组解析,对于发酵过程白酒高品温大曲重要风味物质的产生机理的研究及提高大曲品质具有重要意义。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. .
发明内容Contents of the invention
本发明所要解决的技术问题是现有白酒大曲DNA的提取方法不能满足发酵过程高品温大曲宏基因组测序需求的问题。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.
本发明解决其技术问题所采用的技术方案是:白酒发酵过程高品温大曲总DNA的提取方法,包括如下步骤: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.取白酒发酵过程高品温大曲,快速粉碎,于-80℃冷冻保藏备用;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.将50-100g步骤a备用的大曲与含无菌PBS缓冲液混合均匀后装入无菌离心管中离心,取上清液;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.将步骤b得到的上清液装入无菌离心管中离心,除去上清液,得到菌体;c. Put the supernatant obtained in step b into a sterile centrifuge tube and centrifuge, remove the supernatant to obtain bacterial cells;
d.将1mL PBS缓冲液与菌体混匀并装入无菌离心管中,再加入10μL的溶壁酶、溶菌酶和蛋白酶K,最后加入玻璃珠进行研磨;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.采用FastDNA Spin Kit for Soil试剂盒去除研磨后菌体的蛋白,洗涤,得到白酒发酵过程高品温大曲总DNA。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.
上述步骤a中,粉碎大曲粗细为过20目筛。In the above step a, the size of the crushed Daqu should be passed through a 20-mesh sieve.
上述步骤b中,无菌PBS缓冲液与步骤a备用的大曲质量比为1:8-10。In the above step b, the mass ratio of sterile PBS buffer to the Daqu prepared in step a is 1:8-10.
上述步骤b中,离心的转速为1500-1800rpm,离心时间为5min。In the above step b, the centrifugation speed is 1500-1800 rpm, and the centrifugation time is 5 minutes.
上述步骤c中,离心的转速为12000rpm,离心时间为5min。In the above step c, the centrifugation speed is 12000 rpm and the centrifugation time is 5 minutes.
上述步骤d中,溶壁酶的加入量为20g/L,溶菌酶的加入量为50g/L,蛋白酶K的加入量为10g/L。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.
上述步骤d中,玻璃珠为直径0.2-1mm的无菌玻璃珠,加入量为0.2-0.5g。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.
上述步骤d中,研磨的方法为将无菌离心管在研磨仪研磨10s后于-20℃冷藏2min,然后继续研磨10s。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.
本发明的有益效果是:本发明研究了一种发酵过程高品温大曲总DNA高效提取方法,通过创新的DNA前处理提取法结合低温预冷研磨法,最后经过沉淀洗涤收集,即得发酵过程高品温大曲微生物的总DNA。本发明的提取方法操作方便、快速、高效;提取过程简单、易操作,克服了现有DNA提取方法不能提取发酵过程高品温大曲总DNA的缺点;且操作快速,可在2h内完成大曲总DNA的提取;提取的白酒大曲浓度高达到30-100ng/L,且纯度高,A260/A280值为1.8-2.0。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.
本发明提供的总DNA的高效提取方法可应用于发酵过程高品温大曲样品,解决了发酵过程高品温大曲微生物浓度低,总DNA难提取的问题。本发明从发酵过程高品温大曲中首次分离得到浓度高、纯度高的总DNA,可直接用于宏基因组分析。本发明可为发酵过程高品温大曲中微生物的宏基因组信息分析提供技术支撑,对于大曲发酵过程重要风味物质产生机理的研究及提高大曲品质具有重要意义。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.
附图说明Description of drawings
图1为本发明实施例1-2与对比例1-2提取的总DNA浓度柱状图。Figure 1 is a histogram of the total DNA concentration extracted in Example 1-2 and Comparative Example 1-2 of the present invention.
具体实施方式Detailed ways
本发明的技术方案,具体可以按照以下方式实施。The technical solution of the present invention can be implemented specifically in the following manner.
所述白酒发酵过程高品温大曲总DNA的提取方法,具体步骤如下:The specific steps for extracting total DNA from high-grade Wen Daqu during the liquor fermentation process are as follows:
(1)取白酒发酵过程高品温大曲样品,快速粉碎,-80℃冷冻保藏备用;(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)称取粉碎的大曲,加入到含无菌PBS缓冲液三角瓶中,勤摇匀30min;(2) Weigh the crushed Daqu, add it to an Erlenmeyer flask containing sterile PBS buffer, and shake frequently for 30 minutes;
(3)将溶液转移至50mL无菌离心管,1500-1800rpm离心5min;(3) Transfer the solution to a 50mL sterile centrifuge tube and centrifuge at 1500-1800rpm for 5 minutes;
(4)将上清液转移至新的无菌离心管中,12000rpm离心5min,弃上清,直至收集完所有PBS大曲溶液,得到菌体;(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)加入1mL PBS缓冲液至菌体中,溶解菌体并转移至2mL无菌管中,再加入加入10μl的溶壁酶(20g/L)、溶菌酶(50g/L)和蛋白酶K(10g/L),并加入玻璃珠0.2g;(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)将2mL离心管在研磨仪研磨10s,-20℃冷藏2min,继续研磨10s,重复研磨一次;(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)后续结合FastDNA Spin Kit for Soil试剂盒去蛋白,洗涤,即得总DNA。(7) Subsequently combine the FastDNA Spin Kit for Soil kit to remove protein and wash to obtain the total DNA.
其中,上述步骤(1)粉碎大曲粗细为过20目筛;Wherein, the above-mentioned step (1) crushes Daqu to a thickness of passing through a 20-mesh sieve;
其中,上述步骤(2)称取大曲质量为50-100g;Among them, the above-mentioned step (2) weighs the mass of Daqu to be 50-100g;
其中,上述步骤(2)加入PBS缓冲液搅拌,PBS缓冲液与大曲质量比为1:8-10;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;
其中,上述步骤(5)溶解菌体需要用无菌吸头充分吹打混匀,加入的玻璃珠为无菌且直径为0.2-1mm;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;
其中,上述步骤(6)研磨时间为10s,防止DNA在长时间振荡而产生高温;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;
其中,上述步骤(6)研磨一次后需在-20℃条件下冷藏2min,以降低离心管温度,防止继续振荡产生高温使得DNA被断裂为小片段;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;
其中,上述所有操作步骤,均使用无菌材料;Among them, all the above operation steps use sterile materials;
本发明还提供了上述白酒发酵过程高品温大曲总DNA快速提取的方法在发酵过程高品温大曲总DNA提取中的用途。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.
下面通过实际的例子对本发明的技术方案和效果做进一步的说明。The technical solutions and effects of the present invention will be further described below through practical examples.
实施例Example
实施例1发酵10天、温度为60℃的五粮液包包曲的总DNA提取Example 1 Extraction of total DNA from Wuliangye Baobaoqu fermented for 10 days at a temperature of 60°C
1、取样1. Sampling
从发酵10天的3个五粮液包包曲曲房中,每个曲房随机抽取1块温度为60℃左右的五粮液包包曲样品,装入无菌袋中,用粉碎机粉碎至过20目筛大小,装在无菌袋中,迅速放置于-80℃冰箱中保存备用。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、总RNA的提取2. Extraction of total RNA
提取步骤为:The extraction steps are:
(1)称取50g粉碎的发酵过程高品温大曲,加入到含无菌PBS缓冲液三角瓶中,勤摇匀30min;(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)将上述溶液转移至50mL无菌离心管,1800rpm离心5min;(2) Transfer the above solution to a 50mL sterile centrifuge tube and centrifuge at 1800rpm for 5 minutes;
(3)将上清液转移至新的无菌离心管中,12000rpm离心5min,弃上清,得到菌体;(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)重复上述步骤(2)和(3)共计6次,收集完所有菌体;(4) Repeat the above steps (2) and (3) for a total of 6 times to collect all the bacteria;
(5)加入1mLPBS缓冲液至上述菌体中,用无菌吸头吹打使得菌体充分溶于缓冲液中,并转移至2mL无菌管中,再加入10μL溶壁酶、溶菌酶和蛋白酶K,并加入直径为0.2-1mm的玻璃珠;(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)将2mL离心管在研磨仪研磨10s,-20℃冷藏2min,继续研磨10s,重复研磨一次;(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)后续结合FastDNA Spin Kit for Soil试剂盒去蛋白,加60μL洗脱液进行洗涤,即得总DNA。(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、总DNA的检测3. Detection of total DNA
吸取1μL提取得到的总DNA,使用Thermo scitific公司的Nanodrop one测量其纯度和浓度,结果见表1和图1,可知,发酵10天温度为60℃的五粮液包包曲总DNA浓度为30-100ng/μL,A260/A280值为1.8-2.0,表明DNA纯度和浓度均高。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.
实施例2发酵15天、温度为59℃的五粮液包包曲的总DNA提取Example 2 Extraction of total DNA from Wuliangye Baobaoqu fermented for 15 days at a temperature of 59°C
1、取样1. Sampling
从发酵15天的3个五粮液包包曲曲房中,每个曲房随机抽取1块温度为59℃左右的五粮液包包曲样品,装入无菌袋中,用粉碎机粉碎至过20目筛大小,装在无菌袋中,迅速放置于-80℃冰箱中保存备用。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、总DNA的提取,具体操作同实施例1。2. Extraction of total DNA, the specific operation is the same as in Example 1.
3、总DNA的检测3. Detection of total DNA
吸取1μL提取得到的总DNA,使用Thermo scitific公司的Nanodrop one测量其纯度和浓度,结果见表1和图1,可知,发酵15天温度为59℃的五粮液包包曲总DNA浓度为30-100ng/μL,A260/A280值为1.8-2.0,表明DNA纯度和浓度均高。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.
对比例1Comparative example 1
1、取样,同实施例1。1. Sampling is the same as in Example 1.
2、总DNA的提取,提取步骤按照专利CN113215146A所述大曲DNA提取步骤进行DNA提取。2. Extraction of total DNA. The extraction steps are based on the Daqu DNA extraction steps described in patent CN113215146A.
3、总DNA的检测3. Detection of total DNA
吸取1μL提取得到的总DNA,使用Thermo scitific公司的Nanodrop one测量其纯度和浓度,结果见表1和图1,可知,按照此方法提取的发酵10天温度为60℃的五粮液包包曲总DNA浓度小于10ng/μL,A260/A280值为不在1.8-2.0之间,表明DNA浓度低且纯度不高,不能满足宏基因组测序要求。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.
对比例2Comparative example 2
1、取样,取样同实施例2。1. Sampling, sampling is the same as in Example 2.
2、总DNA的提取,提取步骤按照专利CN113215146A所述大曲DNA提取步骤进行前处理,结合FastDNA Spin Kit for Soil试剂盒进行DNA提取。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、总DNA的检测3. Detection of total DNA
吸取1μL提取得到的总DNA,使用Thermo scitific公司的Nanodrop one测量其纯度和浓度,结果见表1和图1,可知,按照此方法提取的发酵15天温度为59℃的五粮液包包曲总DNA浓度小于10ng/μL,A260/A280值为不在1.8-2.0之间,表明DNA浓度低且纯度不高,不能满足宏基因组测序要求。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.
表1实施例和对比例包包曲总DNA浓度和纯度检测结果Table 1 Examples and Comparative Examples Baobaoqu total DNA concentration and purity detection results
本发明的发酵过程高品温大曲总DNA提取方法不仅适用于浓香型五粮液包包曲,也适用于各类发酵过程和成品白酒大曲中高品温总DNA提取,发明的方法提取快速,提取的微生物总DNA浓度高、纯度高,能有效提取样品中的微生物。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.
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