Method for Extracting Total Microbial DNA from Moromi

Abstract

The present invention relates to a method for extracting total microbial DNA from moromi: Mix a moromi sample with an aqueous solution of Tween 80, centrifuge the resulting mixture, and collect the moromi precipitate; Under liquid nitrogen conditions, mix the moromi precipitate and the activated cation exchange resin and grind, and collect the powder; Mix the powder and the lysozyme buffer solution, incubate to prepare an incubation product; Mix the incubation product and the DNA extraction solution for DNA extraction, centrifuge the resulting extraction product, and collect the supernatant; Mix the supernatant and ethanol for DNA precipitation, centrifuge the resulting precipitate product, collect the DNA precipitate, and wash to obtain total microbial DNA. By using the DNA extraction process of the present invention, total microbial DNA can be extracted from the high-salt and dilute-state soy sauce fermented moromi with high salt content and complex components, and the quality of the total microbial DNA is high, including high purity and good integrity, and can meet the sequencing requirements. The extraction method of the present invention is simple, safe, and can be quickly operated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a method for extracting total microbial DNA from moromi. Background Art

[0002] The history of soy sauce brewing in China is long. Among them, the soy sauce brewed by the high-salt dilute-state process has a strong flavor and delicious taste, which is the main direction of future soy sauce development. The open koji-making method makes the microbial sources of the mature koji and moromi very extensive, and the instability of the microbial composition will lead to fluctuations in the quality of moromi, ultimately affecting the stability of soy sauce production and the quality of finished products. Therefore, it is particularly important to clarify the microbial community structure in moromi for stabilizing and improving the quality of soy sauce and promoting the progress of soy sauce brewing technology.

[0003] At present, the research on moromi microorganisms mainly focuses on the research and analysis of microbial composition through the culturable method. However, the types of microorganisms that can be cultured by the traditional culturable method are very limited, and most unculturable microorganisms are difficult to be discovered. The obtained microbial population cannot accurately represent the microbial community composition of the whole daqu.

[0004] Applying the methods of molecular biology can effectively overcome the deficiencies of traditional culturable techniques. Through amplicon sequencing, the microbial composition of moromi can be obtained quickly and accurately. On this basis, metagenomics can be used to further predict the potential functions of microorganisms in the community. With the continuous progress and development of sequencing technology and omics technology, people have the necessary technical guarantee for the research on the mixed culture fermentation system in moromi. No matter which technology, it is necessary to first obtain total microbial DNA from high-salt dilute-state fermented moromi, and the quality of total microbial DNA determines the reliability of the final result.

[0005] Currently, the commonly used DNA extraction methods mainly include chemical methods and kit methods. Among them, the chemical methods mainly include CTAB method, SDS method, and improved methods based on these methods, such as invention patent CN104560955A; the kit method also uses chemical methods in the sample treatment and DNA extraction stages. The main difference is that it usually uses a purification column to adsorb DNA and then elutes and purifies it, such as patent CN107418952A.

[0006] However, the complex components in high-salt dilute-state fermented moromi pose certain difficulties to the extraction of total microbial DNA. Using the currently commonly used DNA extraction methods, it is impossible to extract high-quality total microbial DNA from high-salt dilute-state fermented moromi to meet the needs of gene sequencing.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The main objective of the embodiments of the present invention is to provide a method for extracting total microbial DNA from moromi, and by using this extraction method, high-quality total microbial DNA can be extracted from the high-salt dilute-state soy sauce fermented moromi.

[0009] The objective of the present invention can be achieved through the following technical solutions:

[0010] The embodiments of the present invention provide a method for extracting total microbial DNA from moromi, characterized in that the extraction method comprises the following steps:

[0011] Mix the moromi sample with an aqueous solution of Tween 80, centrifuge the resulting mixture, and collect the moromi precipitate.

[0012] Under liquid nitrogen conditions, mix the moromi precipitate and the activated cation exchange resin and grind, and collect the powder.

[0013] Mix the powder and the lysozyme buffer solution, incubate, and prepare an incubation product.

[0014] Mix the incubation product and the DNA extraction solution for DNA extraction, centrifuge the resulting extraction product, and collect the supernatant.

[0015] Mix the supernatant and ethanol for DNA precipitation, centrifuge the resulting precipitate product, collect the DNA precipitate, and wash to obtain total microbial DNA.

[0016] In some embodiments of the present invention, the cation exchange resin is a hydrogen-type cation exchange resin or a sodium-type cation exchange resin.

[0017] In some embodiments of the present invention, the steps of activation treatment include: soaking the cation exchange resin in an activator and removing the activator.

[0018] In some embodiments of the present invention, the steps of activation treatment have one or more of the following technical features:

[0019] (1) The activator is an aqueous sodium chloride solution with a sodium chloride content of 5% - 15% (w / v);

[0020] (2) The soaking conditions include: temperature of 15°C - 30°C, time of 0.5 h - 1.5 h;

[0021] (3) The method for removing the activator includes centrifugation, the rotation speed of centrifugation is 8000 rpm - 12000 rpm, and the centrifugation time is 4 min - 6 min.

[0022] In some embodiments of the present invention, the dosage of the cation exchange resin is 10 wt% - 50 wt% of the mass of the moromi sample.

[0023] In some embodiments of the present invention, the average particle diameter of the cation exchange resin is 4 nm - 6 nm.

[0024] In some embodiments of the present invention, the content of Tween 80 in the aqueous Tween 80 solution is 0.2% - 0.8% (w / v).

[0025] In some embodiments of the present invention, the dosage of the aqueous Tween 80 solution corresponding to every 1 g of the moromi sample is 1 mL - 3 mL.

[0026] In some embodiments of the present invention, the conditions for centrifuging the mixture include: the rotation speed is 10000 rpm - 14000 rpm, and the time is 10 min - 20 min.

[0027] In some embodiments of the present invention, the incubation satisfies one or more of the following conditions:

[0028] (1) The dosage of the lysozyme buffer solution corresponding to every 3 g of the powder is 15 mL - 25 mL;

[0029] (2) The incubation temperature is 60°C - 70°C, and the incubation time is 25 min - 35 min.

[0030] In some embodiments of the present invention, the conditions for centrifuging the extraction product include: the rotation speed is 10000 rpm - 14000 rpm, and the time is 5 min - 15 min.

[0031] In some embodiments of the present invention, the conditions for centrifuging the precipitation product include: the rotation speed is 10000 rpm - 14000 rpm, and the time is 5 min - 15 min.

[0032] In some embodiments of the present invention, the cleaning agent used for cleaning the DNA precipitate is an aqueous ethanol solution with an ethanol volume concentration of 65% - 75%.

[0033] In some embodiments of the present invention, the salt content of the moromi sample is 15 wt% - 25 wt%.

[0034] In some embodiments of the present invention, the moromi sample is a high-salt and dilute-state soy sauce fermentation moromi.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention first pre - treats the moromi with an aqueous solution of Tween 80, and then mixes and grinds the pretreated moromi and the activated cation - exchange resin under liquid nitrogen conditions. The obtained powder is incubated with a lysozyme buffer solution and then extracted with a DNA extraction solution, followed by ethanol precipitation and washing, so as to achieve the extraction of total microbial DNA from the moromi. By using the DNA extraction process of the present invention, total microbial DNA can be extracted from the high - salt and dilute - state soy sauce fermented moromi with high salt content and complex components, and the quality of the total microbial DNA is high (including high purity and good integrity), which can meet the sequencing requirements. The extraction method of the present invention is simple, safe and can be quickly operated. Brief Description of the Drawings

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for description in the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0038] Figure 1 It is the agarose gel electrophoresis diagram of total microbial DNA of the moromi samples in the late fermentation stage of the high - salt and dilute - state fermented soy sauce obtained in Example 1 and Comparative Example 1; in the figure, lanes 1 - 5 are the total DNA of 5 parallel moromi samples extracted in Comparative Example 1, lanes 6 - 10 are the total microbial DNA of 5 parallel moromi samples extracted in Example 1, and M is 1000bp ladder;

[0039] Figure 2 It is the agarose gel electrophoresis diagram of the total microbial DNA obtained in Example 2 and Comparative Example 1; in the figure, lane 1 is the total microbial DNA of the moromi sample obtained in Example 2, lane 2 is the total microbial DNA of the moromi sample obtained in Comparative Example 1, and M is 1000bp ladder;

[0040] Figure 3 It is the agarose gel electrophoresis diagram of the PCR products of bacterial 16S rDNA and fungal ITS; in the figure, lanes 1 and 2 are the 16S rDNA of the moromi samples extracted in Example 1 and Example 2 respectively, lanes 5 and 6 are the ITS of the moromi samples extracted in Example 1 and Example 2 respectively, lanes 3, 4 and 7, 8 are the 16S rDNA and ITS of the moromi samples extracted in Comparative Example 1 respectively, and M is 500bp ladder. Detailed Embodiments

[0041] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and not to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the disclosure of the present invention more thoroughly and comprehensively understood. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing the embodiments and examples and are not intended to limit the present invention.

[0043] Terminology

[0044] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0045] The term "and / or", "or / and", "and / or" as used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include any two related listed items, any more related listed items, or the combination of all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are used to connect at least three items, it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example is the technical solution of "A, and / or, B, and / or, C, and / or, D", which includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, includes the combination of any two or any three of A, B, C, D, and also includes the combination of the four items A, B, C, D (that is, the technical solution connected by "logical AND").

[0046] In the present invention, the terms "a plurality of", "a variety of", "multiple times", "multiple elements", etc., unless otherwise specified, refer to a quantity greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0047] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0048] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0049] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0050] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0051] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0052] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0053] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0054] In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, t is an integer selected from 1 to 10, which means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when providing multiple range descriptions of features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0055] The temperature parameter in the present invention, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0056] In the present invention, %(w / w) and wt% both represent weight percentages, %(v / v) refers to volume percentages, and %(w / v) refers to mass-volume percentages.

[0057] All the documents mentioned in the present invention are cited as references in this application, just as if each document is cited separately as a reference. Unless it conflicts with the invention purpose and / or technical solution of this application, the cited documents involved in the present invention are cited for all their contents and all their purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited together. When the present invention involves cited documents, the examples and preferred methods of the relevant technical features cited can also be incorporated as references into this application, but only to the extent that the present invention can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0058] Koji mash refers to a mixture obtained by mixing koji with a large amount of brine to make it in a thick semi-fluid state.

[0059] Currently, the application effect of traditional DNA extraction methods in the extraction of total microbial DNA from high-salt dilute-state fermented koji mash is not good. Traditional DNA extraction methods are, for example:

[0060] A method for separating intracellular and extracellular DNA in sludge and a method for detecting drug-resistant genes carried thereby, as described in Chinese Patent Application for Invention CN113215146A. The core lies in using enzymatic hydrolysis to soften the firm cell wall. Its deficiencies are mainly as follows: 1. The enzymatic treatment process is time-consuming and has cumbersome steps; 2. When extracting DNA in large quantities, the dosage of the enzyme is large and the cost is high; 3. The preference of different enzymes leads to different degrees of enzymatic hydrolysis of the cell walls of different microorganisms, and the DNA of cells with poor enzymatic hydrolysis effect is not fully released; 4. The addition of the enzyme also introduces some exogenous DNA, affecting the purity of the final target DNA.

[0061] A method for extracting the metatranscriptomic RNA of a natural cellulose-utilizing flora, as described in Chinese Invention Patent CN105779442B. The core lies in adding rigid spherical auxiliary grinding agents such as glass beads and ceramic beads, and using the method of bead milling and homogenization to break the cell wall. Due to the excessive shearing force of this method, during the extraction of microbial DNA from moromi samples, a large amount of DNA from koji-making raw materials such as soybeans and wheat mixed in it will be co-extracted, seriously affecting the purity of the total microbial DNA.

[0062] Chinese Patent Application for Invention CN107418952A describes a method for extracting the metagenome of soil microorganisms, which combines enzymatic, chemical, and physical methods to better lyse cells. However, during the DNA purification process, an adsorption column needs to be used, which is essentially the same as the kit method and cannot meet the need for extracting DNA from a large-scale moromi culture system, and the overall experimental steps are relatively cumbersome.

[0063] Chinese Patent Application for Invention CN111875665A describes a method for microparticles for cell disruption and / or recovery of biomolecules, "a method for disrupting cells, including adding negatively charged microparticles to a biological fluid, wherein the negatively charged microparticles are ground polymeric cation exchange resins". However, the inventor found that high-quality total microbial DNA cannot be extracted from high-salt dilute-state fermented soy sauce moromi using its technical solution.

[0064] However, in the actual operation process, the application effect of traditional DNA extraction methods in the extraction of total microbial DNA from high-salt dilute-state fermented moromi is not good. The reasons are mainly as follows:

[0065] 1. The salt content in the high-salt dilute-state soy sauce fermentation system is high. The high-concentration salt ions and various secondary metabolites produced during the fermentation process affect the effect of chemical reagents, and some inhibitors such as (phenolic compounds, heavy metals, etc.) co-extract with DNA;

[0066] 2. Microorganisms in a high-salt environment, such as halotolerant yeasts, have strong high-osmotic pressure resistance and firm cell structures. Whether the cell wall can be effectively broken seriously affects the quality of DNA extraction;

[0067] 3. The genomes of production raw materials such as soybeans and wheat in the moromi seriously interfere with the genomes of target microorganisms, thereby affecting the subsequent use of DNA.

[0068] Based on the above problems, the object of the present invention is to overcome the deficiencies of the prior art and provide a method capable of extracting high-quality total microbial DNA from the moromi of high-salt and dilute-state soy sauce fermentation.

[0069] To this end, the present invention mainly first pretreats the moromi with an aqueous solution of Tween 80, and then mixes and grinds the pretreated moromi and the activated cation exchange resin under liquid nitrogen conditions. The obtained powder is incubated with a lysozyme buffer solution and then extracted with a DNA extraction solution, precipitated with ethanol, and washed, thereby realizing the high-quality (high purity and good integrity) extraction of total microbial DNA in the moromi.

[0070] The moromi of soy sauce fermentation contains a large amount of impurities (metal ions, polysaccharides, phenolic compounds, proteins, cell wall residues, etc.) that are soluble or insoluble in water. The aqueous solution of Tween 80 can well dissolve the water-insoluble impurities and metabolites, and then these impurities and metabolites are removed by centrifuging to remove the supernatant, reducing their impact on the DNA extraction efficiency in the subsequent extraction process.

[0071] By adding a cation exchange resin as an auxiliary grinding body, the present invention can effectively increase the cell wall breaking efficiency of stubborn microorganisms in the moromi, and at the same time reduce the interference of DNA such as soybeans and wheat, and improve the yield of total microbial DNA. In the present invention, the cation exchange resin can be a hydrogen-type cation exchange resin or a sodium-type cation exchange resin, preferably a sodium-type strong cation exchange resin, and more preferably a sodium-type 001*7 cation exchange resin. The reaction group of the hydrogen-type cation exchange resin is a sulfonic acid group, and its exchange ability does not change with the change of pH. The pH of the moromi of high-salt and dilute-state soy sauce fermentation is approximately 4-5, which can ensure that impurities such as proteins carry a positive charge and are tightly bound to the cation exchange resin, thereby ensuring the purification effect. The addition amount of the cation exchange resin is about 1 / 10-5 / 10 (w / w) of the moromi. During the grinding process, it should be noted to add liquid nitrogen in time, otherwise the sample will absorb moisture in the air and become viscous, affecting the grinding efficiency. The pestle should be quickly ground in one direction, and when no obvious large particles can be observed in the sample, it can be quickly transferred to the lysis buffer solution. After grinding, the moromi and the cation exchange resin simultaneously become a powdery mixture, and some of the contents after cell wall breaking can be adsorbed by the charged cation exchange resin. During the incubation process, it should be gently pipetted and mixed with a pipette tip in time to make the reaction more complete.

[0072] For grinding, the inventors of the present invention have tried to add rigid glass beads during liquid nitrogen grinding to increase mechanical shear force, but the final extraction effect is not good. The main reason is that commercial glass beads have a unified specification, and different sizes have preferences for the cell wall breaking effects of different microorganisms, and the cell wall breaking effect on large particle raw materials in moromi is more obvious. And the cation exchange resin gradually breaks during grinding, and there are resins of different sizes in the mixture, which generate shear force on the cell walls of different microorganisms. In addition, compared with the inert auxiliary grinding agents (such as ceramic beads, glass beads, silicon beads, etc.) of other DNA extraction methods (such as the method described in CN105779442B), the addition of cation exchange resin has the following irreplaceable advantages:

[0073] 1. The cation exchange resin in the form of powder after grinding can better exchange the salt ions in the system and reduce its interference with DNA extraction;

[0074] 2. Since the cation exchange resin itself has a charge, it has a certain adsorption effect on impurities such as polysaccharides, proteins, and polyphenols in the moromi system, making the extracted DNA purer;

[0075] 3. The cation exchange resin provides appropriate shear force in the specific scenario of extracting moromi microbial DNA, ensuring good separation of microbial cells from koji-making raw materials. While inert auxiliary grinding agents such as ceramic beads, glass beads, and silicon beads have strong rigidity, and the shear force on koji-making raw materials (soybeans, wheat) during grinding is too large, resulting in a large amount of their DNA being co-extracted in the subsequent process, causing serious interference to the target microbial DNA.

[0076] Furthermore, the inventors have also tried to directly grind the pretreated moromi under liquid nitrogen (conventional auxiliary grinding agents can also be added to provide shear force), and then react it under the ground cation exchange resin. In this method, the ground resin only acts as an adsorbent for some impurities during DNA extraction, and the phenomenon of serious co-extraction of koji-making raw material DNA has not been solved, and the quality of the obtained DNA is not sufficient for subsequent sequencing analysis.

[0077] In addition, the inventors have also tried to directly add the ground powder to the DNA extraction solution for extraction without sufficient incubation in the lysing buffer in step 3, and finally only trace amounts of DNA are contained in the eluate obtained. The main purpose of step 3 is to use ionic surfactants such as (sodium dodecyl sulfate) SDS to dissolve the cell membrane and nuclear membrane proteins, break the cell membrane and nuclear membrane, and fully release DNA.

[0078] The embodiment of the present invention provides a method for extracting total DNA of microorganisms in moromi, and the extraction method includes the following steps:

[0079] Mix the moromi sample with an aqueous solution of Tween 80, centrifuge the obtained mixture, and collect the moromi precipitate;

[0080] Under liquid nitrogen conditions, mix the moromi precipitate and the activated cation exchange resin and grind them, and collect the powder.

[0081] Mix the powder and the lysozyme buffer solution, incubate, and prepare an incubation product.

[0082] Mix the incubation product and the DNA extraction solution for DNA extraction, centrifuge the obtained extraction product, and collect the supernatant.

[0083] Mix the supernatant and ethanol for DNA precipitation, centrifuge the obtained precipitation product, collect the DNA precipitate, and wash it to obtain the total microbial DNA.

[0084] In some embodiments, the cation exchange resin is a hydrogen-type cation exchange resin or a sodium-type cation exchange resin.

[0085] In some embodiments, the steps of activation treatment include: soaking the cation exchange resin in an activator and removing the activator.

[0086] In some embodiments, the steps of activation treatment have one or more of the following technical features:

[0087] (1) The activator is an aqueous sodium chloride solution with a sodium chloride content of 5%-15% (w / v);

[0088] (2) The soaking conditions include: the temperature is 15°C - 30°C, and the time is 0.5 h - 1.5 h;

[0089] (3) The method for removing the activator includes centrifugation, the centrifugation speed is 8000 rpm - 12000 rpm, and the centrifugation time is 4 min - 6 min.

[0090] In the activator, the sodium chloride content (%(w / v)) is, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

[0091] The soaking temperature (°C) is, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and the soaking time (h) is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5.

[0092] The centrifugation speed (rpm) for removing the activator is, for example, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, 12500, and the centrifugation time (min) is, for example, 4, 4.5, 5, 5.5, 6, 6.5.

[0093] In some embodiments, the dosage of the cation exchange resin is 10 wt% - 50 wt% of the mass of the moromi sample, such as 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, 50 wt%.

[0094] In some embodiments of the present invention, the average particle diameter of the cation exchange resin is 4 nm - 6 nm, such as 4 nm, 4.5 nm, 5 nm, 5.5 nm, 6 nm.

[0095] In some embodiments of the present invention, the content of Tween 80 in the Tween 80 aqueous solution is 0.2% - 0.8% (w / v), such as 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%.

[0096] In some embodiments of the present invention, the dosage of the Tween 80 aqueous solution corresponding to every 1 g of the moromi sample is 1 mL - 3 mL, such as 1 mL, 1.2 mL, 1.4 mL, 1.6 mL, 1.8 mL, 2 mL, 2.2 mL, 2.4 mL, 2.6 mL, 2.8 mL, 3 mL.

[0097] In some embodiments of the present invention, the conditions for centrifuging the mixture include: the rotation speed is 10000 rpm - 14000 rpm, and the time is 10 min - 20 min. The rotation speed (rpm) of centrifugation is, for example, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, and the time (min) of centrifugation is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0098] In some embodiments of the present invention, the incubation satisfies one or more of the following conditions:

[0099] (1) The dosage of the lysozyme buffer solution corresponding to every 3 g of the powder is 15 mL - 25 mL; and,

[0100] (2) The incubation temperature is 60 °C - 70 °C, and the incubation time is 25 min - 35 min.

[0101] The dosage (mL) of the lysozyme buffer solution corresponding to every 3 g of the powder is, for example, 15, 17, 19, 20, 21, 23, 25.

[0102] The incubation temperature (°C) is, for example, 60, 62, 64, 66, 68, 70, and the incubation time (min) is, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35.

[0103] In some embodiments of the present invention, the conditions for centrifuging the extraction product include: a rotation speed of 10,000 rpm - 14,000 rpm and a time of 5 min - 15 min. The rotation speed (rpm) of centrifugation is, for example, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, and the time (min) of centrifugation is, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

[0104] In some embodiments of the present invention, the conditions for centrifuging the precipitation product include: a rotation speed of 10,000 rpm - 14,000 rpm and a time of 5 min - 15 min. The rotation speed (rpm) of centrifugation is, for example, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, and the time (min) of centrifugation is, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.

[0105] In some embodiments of the present invention, the cleaning agent used for cleaning the DNA precipitate is an aqueous ethanol solution with an ethanol volume concentration of 65% - 75%. The ethanol volume concentration (%) in the aqueous ethanol solution is, for example, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75.

[0106] In some embodiments of the present invention, the salt content of the moromi sample is 15 wt% - 25 wt%, for example, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25 wt%.

[0107] In some embodiments of the present invention, the moromi sample is a high-salt and dilute-state soy sauce fermentation moromi. Specific embodiments

[0109] The following will describe the implementation scheme of the present invention in detail in combination with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, the guidance given in the present invention is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0110] In the following specific embodiments, for the measurement parameters of the raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0111] 1. Lysozyme buffer: 50 mM Tris-HCl, 50 mM EDTA, 3% SDS, pH 8.0. Store and use after autoclaving at high temperature and high pressure.

[0112] 2. DNA extraction solution: Phenol:Chloroform:Isoamyl alcohol (25:24:1). Gently invert and mix well (chloroform and isoamyl alcohol are prepared in advance according to the ratio).

[0113] 3. The cation exchange resin needs to be soaked in 10% (w / v) sodium chloride.

[0114] Example 1. Method for extracting total microbial DNA from fermented mash of high-salt dilute-state soy sauce

[0115] An example of the method for extracting total microbial DNA from fermented mash of high-salt dilute-state soy sauce in the present invention includes the following steps:

[0116] a. Pretreatment of moromi sample

[0117] 1. Weigh 10 g of fermented mash of high-salt dilute-state soy sauce with a salt content of 18 wt% and dissolve it in 20 mL of 0.5% (w / v) Tween80 aqueous solution. Centrifuge at 12000 rpm for 15 min, then discard the supernatant and collect the precipitate.

[0118] b. Activation of cation exchange resin

[0119] 2. Add 10 mL of activator to each gram of resin. The activator is 10% (w / v) NaCl aqueous solution. Treat it at room temperature (15 - 30 °C) for 1 h. After activation, centrifuge at 10000 rpm for 5 min and remove the supernatant to obtain the activated resin. Among them, the resin selected is sodium-type 001*7 cation exchange resin with an average particle diameter of 5 nm.

[0120] c. Grinding

[0121] 3. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen, add 1 g of activated cation exchange resin and mix it with the precipitate. Use a pestle to gradually grind the precipitate and the activated cation exchange resin into powder, continuously adding liquid nitrogen to keep it at a low temperature during the process until the grinding product is in a light yellow powder form.

[0122] d. Extraction of total DNA

[0123] 4. Weigh 3 g of the powder and add 20 mL of lysing buffer preheated at 65 °C.

[0124] 5. Incubate at 65 °C for 30 min and gently mix every 5 min with a pipette tip without the tip end.

[0125] 6. Add an equal volume of DNA extraction solution and centrifuge at 12,000 rpm for 10 min.

[0126] 7. Carefully aspirate the supernatant and transfer it to a new centrifuge tube. Repeat step 6 once.

[0127] e. Washing of DNA precipitate

[0128] 8. Take 10 mL of the supernatant and add 25 mL of anhydrous ethanol pre-cooled at -20 °C.

[0129] 9. Precipitate DNA at -20 °C for 30 min, centrifuge at 12,000 rpm and 4 °C for 10 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0130] 10. Discard the supernatant, add 20 mL of 70% (v / v) ethanol solution, and gently pipette the precipitate with a pipette tip to make it float.

[0131] 11. Centrifuge at 12,000 rpm for 8 min and repeat step 10 once.

[0132] 12. Centrifuge at 12,000 rpm for 8 min and discard the supernatant.

[0133] f. Dissolution of DNA

[0134] 13. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol, and add 0.5 mL of sterile water to dissolve the precipitate.

[0135] Using the method of this example, the total DNA of the mash microorganisms during the fermentation of high-salt dilute-state soy sauce was obtained, and the quality of the total DNA was detected by agarose gel electrophoresis. The electrophoresis patterns of 5 parallel samples of high-salt dilute-state soy sauce fermentation mash are shown in Figure 1 lanes 6-10 in. The results show that the band brightness is high and there is no protein and phenol contamination. The absorbance ratio of A260 / A280 measured by a UV spectrophotometer is 1.92, and the concentration is 57.5 μg / mL (taking the total DNA of the parallel sample of the high-salt dilute-state soy sauce fermentation mash corresponding to Figure 1 lane 6 as an example). The electrophoresis results of the PCR products of 16S rDNA and ITS (see Figure 3 lanes 1, 5) show that the corresponding bands are clearly visible, meeting the sample quality requirements for genome sequencing.

[0136] Example 2. Method for Extracting Total DNA of Microorganisms in High-Salt Dilute-State Soy Sauce (with a Salt Content of 15 wt%) Fermentation Mash

[0137] a. Pretreatment of moromi sample

[0138] 1. Weigh 10 g of high-salt dilute-state soy sauce fermentation mash with a salt content of 15 wt%, dissolve it in 20 mL of 0.5% (w / v) Tween 80 aqueous solution, centrifuge at 12000 rpm for 15 min, discard the supernatant, and collect the precipitate.

[0139] b. Activation of cation exchange resin

[0140] 2. Add 10 mL of activator to each gram of resin. The activator is 10% (w / v) NaCl aqueous solution. Treat it at room temperature (15 - 30 °C) for 1 h. After activation, centrifuge at 10000 rpm for 5 min, remove the supernatant, and obtain the activated resin. Among them, the resin selected is sodium-type 001*7 cation exchange resin, and its average particle diameter is 5 nm.

[0141] c. Grinding

[0142] 3. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen, add 1 g of activated cation exchange resin and mix it with the precipitate. Use a pestle to gradually grind the precipitate and the activated cation exchange resin into powder. During this process, continuously add liquid nitrogen to maintain low temperature until the grinding product becomes a light yellow powder.

[0143] d. Extraction of total DNA

[0144] 4. Weigh 3 g of the powder and add 20 mL of lysing buffer preheated at 65 °C.

[0145] 5. Incubate at 65 °C for 30 min, and gently mix it with a pipette tip without a tip every 5 min.

[0146] 6. Add an equal volume of DNA extraction solution and centrifuge at 12000 rpm for 10 min.

[0147] 7. Carefully aspirate the supernatant and transfer it to a new centrifuge tube, and repeat step (6) once.

[0148] e. Washing of DNA precipitate

[0149] 8. Take 10 mL of the supernatant and add 25 mL of anhydrous ethanol pre-cooled at -20 °C.

[0150] 9. Precipitate DNA at -20 °C for 30 min, centrifuge at 12000 rpm and 4 °C for 10 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0151] 10. Discard the supernatant, add 20 mL of 70% (v / v) ethanol solution, and gently pipette the precipitate with a pipette tip to make it float.

[0152] 11. Centrifuge at 12000 rpm for 8 min and repeat step (10) once.

[0153] 12. Centrifuge at 12,000 rpm for 8 min and discard the supernatant.

[0154] f. Dissolution of DNA

[0155] 13. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol, and add 0.5 mL of sterile water to dissolve the precipitate.

[0156] Using the method of this example, the total DNA of the mash microorganisms during the fermentation of high-salt dilute-state soy sauce was obtained. The quality of the total DNA was detected by agarose gel electrophoresis. See Figure 2 , and the results showed that the band brightness was high, without protein and phenol contamination. The absorbance ratio of A260 / A280 measured on a UV spectrophotometer was 1.79, and the concentration was 41.3 μg / mL. The electrophoresis results of the PCR products of 16S rDNA and ITS (see Figure 3 lanes 2 and 6) showed that the corresponding bands were clearly visible, meeting the sample quality requirements for genome sequencing.

[0157] Example 3. Method for Extracting Total DNA of Mash Microorganisms in the Fermentation of High-Salt Dilute-State Soy Sauce

[0158] a. Pretreatment of moromi sample

[0159] 1. Weigh 10 g of high-salt dilute-state soy sauce fermentation mash with a salt content of 15 wt% and dissolve it in 30 mL of 0.2% (w / v) Tween 80 aqueous solution. After centrifuging at 14,000 rpm for 10 min, discard the supernatant and collect the precipitate.

[0160] b. Activation of cation exchange resin

[0161] 2. Add 10 mL of activator to each gram of resin. The activator is 15% (w / v) NaCl aqueous solution. Treat it at room temperature (15 - 30 °C) for 0.5 h. After activation, centrifuge at 8,000 rpm for 4 min and remove the supernatant to obtain the activated resin. Among them, the resin is a hydrogen-type cation exchange resin with an average particle diameter of 4 nm.

[0162] c. Grinding

[0163] 3. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen, add 3 g of activated cation exchange resin and mix it with the precipitate. Use a pestle to gradually grind the precipitate and the activated cation exchange resin into a powder, continuously adding liquid nitrogen to keep it at a low temperature during the process until the grinding product is in a light yellow powder form.

[0164] d. Extraction of total DNA

[0165] 4. Weigh 3 g of the powder and add 15 mL of lysing buffer preheated to 60 °C.

[0166] 5. Incubate at 60 °C for 25 min and gently mix every 5 min with a pipette tip without the tip end.

[0167] 6. Add an equal volume of DNA extraction solution and centrifuge at 10,000 rpm for 15 min.

[0168] 7. Carefully aspirate the supernatant and transfer it to a new centrifuge tube. Repeat step 6 once.

[0169] e. Washing of DNA precipitate

[0170] 8. Take 10 mL of the supernatant and add 25 mL of absolute ethanol pre-cooled to -20 °C.

[0171] 9. Precipitate DNA at -20 °C for 30 min and centrifuge at 10,000 rpm at 4 °C for 15 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0172] 10. Discard the supernatant, add 20 mL of 65% (v / v) ethanol solution, and gently pipette the precipitate to make it float.

[0173] 11. Centrifuge at 10,000 rpm for 5 min and repeat the operation in step 10 once.

[0174] 12. Centrifuge at 10,000 rpm for 5 min and discard the supernatant.

[0175] f. Dissolution of DNA

[0176] 13. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol, and add 0.5 mL of sterile water to dissolve the precipitate.

[0177] Using the method of this example, the total DNA of the microorganisms in the moromi during the fermentation of high-salt liquid-state soy sauce was obtained. The quality of the total DNA was detected by agarose gel electrophoresis. The results showed that the band brightness was high and there was no protein and phenol contamination. The absorbance ratio of A260 / A280 measured by an ultraviolet spectrophotometer was 1.75, and the concentration was 40.8 μg / mL. The electrophoresis results of the PCR products of 16S rDNA and ITS showed that the corresponding bands were clearly visible, meeting the sample quality requirements for genome sequencing.

[0178] Example 4. Method for Extracting Total DNA of Microorganisms in Moromi during Fermentation of High-Salt Liquid-State Soy Sauce

[0179] An example of the method for extracting total DNA of microorganisms from the moromi during the fermentation of high-salt liquid-state soy sauce in the present invention includes the following steps:

[0180] a. Pretreatment of moromi sample

[0181] 1. Weigh 10 g of a high-salt dilute-state soy sauce fermentation mash with a salt content of 25 wt%, dissolve it in 10 mL of a 0.8% (w / v) Tween 80 aqueous solution, centrifuge at 10,000 rpm for 20 min, discard the supernatant, and collect the precipitate.

[0182] b. Activation of cation exchange resin

[0183] 2. Add 10 mL of an activator to each gram of resin. The activator is a 5% (w / v) NaCl aqueous solution. Treat it at room temperature (15 - 30 °C) for 1.5 h. After activation, centrifuge at 12,000 rpm for 6 min, remove the supernatant, and obtain the activated resin. Among them, the resin selected is a hydrogen-type cation exchange resin with an average particle diameter of 6 nm.

[0184] c. Grinding

[0185] 3. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen, add 5 g of the activated cation exchange resin and mix it with the precipitate. Use a pestle to gradually grind the precipitate and the activated cation exchange resin into a powder. During this process, continuously add liquid nitrogen to maintain a low temperature until the grinding product is in a light yellow powder form.

[0186] d. Extraction of total DNA

[0187] 4. Weigh 3 g of the powder and add 25 mL of a lysing buffer pre-heated to 70 °C.

[0188] 5. Incubate at 70 °C for 35 min and gently mix every 5 min with a pipette tip without the tip end.

[0189] 6. Add an equal volume of DNA extraction solution and centrifuge at 14,000 rpm for 5 min.

[0190] 7. Carefully aspirate the supernatant and transfer it to a new centrifuge tube. Repeat step 6 once.

[0191] e. Washing of DNA precipitate

[0192] 8. Take 10 mL of the supernatant and add 25 mL of anhydrous ethanol pre-cooled to -20 °C.

[0193] 9. Precipitate DNA at -20 °C for 30 min, centrifuge at 14,000 rpm and 4 °C for 5 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0194] 10. Discard the supernatant, add 20 mL of a 75% (v / v) ethanol solution, and gently pipette the precipitate with a pipette tip to make it float.

[0195] 11. Centrifuge at 14,000 rpm for 5 min and repeat step 10 once.

[0196] 12. Centrifuge at 14,000 rpm for 5 min and discard the supernatant.

[0197] f. Dissolution of DNA

[0198] 13. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol, and add 0.5 mL of sterile water to dissolve the precipitate.

[0199] Using the method of this example, the total DNA of the mash microorganisms in the high-salt liquid-state soy sauce fermentation process was obtained. The quality of the total DNA was detected by agarose gel electrophoresis. The results showed that the band brightness was high and there was no protein and phenol contamination. The absorbance ratio of A260 / A280 measured by a UV spectrophotometer was 1.82, and the concentration was 46.2 μg / mL. The electrophoresis results of the PCR products of 16S rDNA and ITS showed that the corresponding bands were clearly visible, meeting the sample quality requirements for genome sequencing.

[0200] Comparative Example 1. Method for extracting total DNA of mash microorganisms in high-salt liquid-state soy sauce fermentation without adding cation exchange resin relative to Example 1

[0201] a. Pretreatment of mash sample

[0202] 1. Weigh 10 g of high-salt liquid-state soy sauce fermentation mash with a salt content of 18 wt% and dissolve it in 20 mL of 0.5% Tween 80 aqueous solution. After centrifuging at 12,000 rpm for 15 min, discard the supernatant and collect the precipitate.

[0203] b. Grinding

[0204] 2. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen and directly grind the precipitate into powder with a pestle. During this process, continuously add liquid nitrogen to keep it at a low temperature until the precipitate becomes a pale yellow powder.

[0205] c. Extraction of total DNA

[0206] 3. Take 3 g of the powder and add 20 mL of lysing buffer preheated at 65 °C.

[0207] 4. Incubate at 65 °C for 30 min and gently mix every 5 min with a pipette tip without the tip.

[0208] 5. Add an equal volume of DNA extraction solution and centrifuge at 12,000 rpm for 10 min.

[0209] 6. Carefully aspirate the supernatant (do not aspirate the white precipitate in the middle layer) and repeat the operation in step 5 once.

[0210] d. Washing of DNA precipitate

[0211] 7. Take 10 mL of the supernatant and add 25 mL of anhydrous ethanol pre-cooled at -20°C.

[0212] 8. Precipitate DNA at -20°C for 30 min, centrifuge at 12000 rpm and 4°C for 10 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0213] 9. Discard the supernatant, add 20 mL of 70% (v / v) ethanol solution, and gently pipette the precipitate with a pipette tip to make it float.

[0214] 10. Centrifuge at 12000 rpm for 8 min, and repeat the operation in step 9 once.

[0215] 11. Centrifuge at 12000 rpm for 8 min and discard the supernatant.

[0216] e. Dissolution of DNA

[0217] 12. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol, and add 0.5 mL of sterile water to dissolve the precipitate.

[0218] Using the method of this comparative example, the total microbial DNA of the moromi without adding cation exchange resin during grinding was obtained. During the implementation of this comparative example, since cation exchange resin was not added during the grinding process, the effect of breaking the microbial cell wall by subsequent liquid nitrogen grinding was poor, and the impurities were not completely removed. Through quality detection by agarose gel electrophoresis, the results (see Figure 2 Lane 2) showed that the corresponding DNA brightness was low, and the diffuse total DNA band could barely be seen. The concentration measured by the ultraviolet spectrophotometer was 14.4 μg / mL, and the absorbance ratio of A260 / A280 measured by the ultraviolet spectrophotometer was 2.27, indicating a relatively high RNA residue. In addition, the main bright band was small, and the band at the bottom of the lane was obvious, indicating that DNA degradation was relatively serious. The 16S rDNA electrophoresis results (see Figure 3 Lanes 3 and 4) showed that the corresponding bands were relatively dim. It should be particularly noted that the ITS electrophoresis results (see Figure 3 Lanes 7 and 8) showed that the corresponding band size was about 750 bp, which was larger than the general fungal ITS size, indicating that the main source of this band was raw materials such as soybeans and wheat.

[0219] It can be seen that under the condition of not using cation exchange resin for grinding, the total DNA content and quality extracted from the high-salt dilute-state soy sauce fermentation moromi by using conventional liquid nitrogen grinding combined with the SDS method are both low.

[0220] Comparative Example 2. Method for Extracting Total Microbial DNA of High-Salt Dilute-State Soy Sauce Fermentation Moromi with the Resin Not Activated as Compared with Example 1

[0221] a. Pretreatment of moromi sample

[0222] 1. Weigh 10 g of the high-salt dilute-state soy sauce fermentation mash with 18 wt% salt content and dissolve it in 20 mL of 0.5% (w / v) Tween 80 aqueous solution. After centrifuging at 12,000 rpm for 15 min, discard the supernatant and collect the precipitate.

[0223] b. Grinding

[0224] 2. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen, add 1 g of unactivated cation exchange resin and mix it with the precipitate. Use a pestle to gradually grind the precipitate and the activated cation exchange resin into a powder, continuously adding liquid nitrogen to maintain a low temperature during the process until the grinding product is in a light yellow powder form.

[0225] c. Extraction of total DNA

[0226] 3. Weigh 3 g of the powder and add 20 mL of lysing buffer pre-heated at 65 °C.

[0227] 4. Incubate at 65 °C for 30 min and gently mix with a pipette tip without the tip every 5 min.

[0228] 5. Add an equal volume of DNA extraction solution and centrifuge at 12,000 rpm for 10 min.

[0229] 6. Carefully aspirate the supernatant and transfer it to a new centrifuge tube, repeating step 5 once.

[0230] d. Washing of DNA precipitate

[0231] 7. Take 10 mL of the supernatant and add 25 mL of anhydrous ethanol pre-cooled at -20 °C.

[0232] 8. Precipitate DNA at -20 °C for 30 min and centrifuge at 12,000 rpm and 4 °C for 10 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0233] 9. Discard the supernatant, add 20 mL of 70% (v / v) ethanol solution, and gently pipette the precipitate with a pipette tip to make it float.

[0234] 10. Centrifuge at 12,000 rpm for 8 min and repeat the operation in step 9 once.

[0235] 11. Centrifuge at 12,000 rpm for 8 min and discard the supernatant.

[0236] e. Dissolution of DNA

[0237] 12. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol, and add 0.5 mL of sterile water to dissolve the precipitate.

[0238] The method of this comparative example is applied to obtain the total DNA of sauce mash microorganisms without activation treatment of the resin. During the implementation process, since the new cation exchange resin is not activated, it contains a small amount of oligomers that do not participate in the polymerization reaction, as well as inorganic impurities such as iron, aluminum, and copper, which affect DNA extraction, resulting in insufficient purity of the extracted DNA. The concentration measured by the ultraviolet spectrophotometer is 42.5 μg / mL, and the absorbance ratio of A260 / A280 measured on the ultraviolet spectrophotometer is 2.33 (normal value is 1.8-2.0), indicating that the DNA purity is significantly lower than that of the embodiment.

[0239] Comparative Example 3: Method for extracting total DNA from fermented soybean paste microorganisms using Tween 20 instead of Tween 80 relative to Example 1

[0240] a. Pretreatment of moromi sample

[0241] 1. Weigh 10 g of high-salt dilute soy sauce fermentation mash with a salt content of 18 wt % and dissolve it in 20 mL of 0.5% (w / v) Tween 20 aqueous solution. Centrifuge at 12000 rpm for 15 min, discard the supernatant, and collect the precipitate.

[0242] b. Activation of cation exchange resin

[0243] 2. Add 10 mL of activator per gram of resin, the activator is 10% (w / v) NaCl, treat at room temperature (15-30°C) for 1 hour, centrifuge at 10000 rpm for 5 minutes after activation, remove the supernatant, and obtain the activated resin.

[0244] c. Grinding

[0245] 3. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen, add 1 g of activated cation exchange resin and mix well with the precipitate, and use a pestle to gradually grind the precipitate and the activated cation exchange resin into powder, while continuously adding liquid nitrogen to keep the temperature low until the ground product becomes a light yellow powder.

[0246] d. Extraction of total DNA

[0247] 4. Weigh 3 g of powder and add 20 mL of lysis buffer preheated at 65°C.

[0248] 5. Incubate at 65°C for 30 min. Mix gently with a sharpened pipette tip every 5 min.

[0249] 6. Add an equal volume of DNA extraction solution and centrifuge at 12000 rpm for 10 min.

[0250] 7. Carefully aspirate the supernatant and transfer it to a new centrifuge tube. Repeat step (6) once.

[0251] e. Washing of DNA precipitate

[0252] 8. Take 10 mL of the supernatant and add 25 mL of anhydrous ethanol pre-cooled at -20 °C.

[0253] 9. Precipitate DNA at -20 °C for 30 min, centrifuge at 12000 rpm and 4 °C for 10 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0254] 10. Discard the supernatant, add 20 mL of 70% (v / v) ethanol solution, and gently pipette the precipitate with a pipette tip to make it float.

[0255] 11. Centrifuge at 12000 rpm for 8 min, and repeat the operation in step (10) once.

[0256] 12. Centrifuge at 12000 rpm for 8 min and discard the supernatant.

[0257] f. Dissolution of DNA

[0258] 13. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol, and add 0.5 mL of sterile water to dissolve the precipitate.

[0259] Using the method of this comparative example, total DNA of the moromi microorganisms was obtained. Tween 20 and Tween 80 are both non-ionic surfactants. The difference is that Tween 20 is more conducive to the dissolution of hydrophilic substances, and Tween 80 is more helpful for the dissolution of hydrophobic substances. The fats and fat-soluble substances (such as fat-soluble proteins) contained in the moromi can be better dissolved in the presence of Tween 80 and separated from the system by centrifugation. Detected by an ultraviolet spectrophotometer, its concentration is 21.6 μg / mL, which is significantly lower than the results of Example 1 and Example 2, and the absorbance ratio of A260 / A280 is 1.54 (lower than the normal level of 1.8 - 2.0), indicating that the residual amount of protein impurities is relatively high, and its quality is not conducive to subsequent gene sequencing.

[0260] Comparative Example 4. Method for extracting total DNA of moromi microorganisms without incubation with lysis buffer relative to Example 1

[0261] a. Pretreatment of moromi sample

[0262] 1. Weigh 10 g of high-salt dilute-state soy sauce fermentation moromi with a salt content of 18 wt% and dissolve it in 20 mL of 0.5% (w / v) Tween 80 aqueous solution. After centrifuging at 12000 rpm for 15 min, discard the supernatant and collect the precipitate.

[0263] b. Activation of cation exchange resin

[0264] 2. Add 10 mL of activator to each gram of resin. The activator is 10% (w / v) NaCl. Treat at room temperature (15 - 30 °C) for 1 h. After activation, centrifuge at 10000 rpm for 5 min, remove the supernatant, and obtain the activated resin.

[0265] c. Grinding

[0266] 3. Transfer the precipitate to a mortar pre-cooled with liquid nitrogen. Add 1 g of activated cation exchange resin and mix well with the precipitate. Gradually grind the precipitate and the activated cation exchange resin into a powder with a pestle, continuously adding liquid nitrogen to maintain low temperature during the process until the grinding product is in a light yellow powder form.

[0267] d. Extraction of total DNA

[0268] 4. Weigh 3 g of the powder. Without incubating with the lysis buffer, directly add an equal volume of DNA extraction solution. Centrifuge at 12000 rpm for 10 min.

[0269] 5. Carefully aspirate the supernatant and transfer it to a new centrifuge tube. Add an equal volume of DNA extraction solution and centrifuge at 12000 rpm for 10 min.

[0270] e. Washing of DNA precipitate

[0271] 6. Take 10 mL of the supernatant and add 25 mL of anhydrous ethanol pre-cooled at -20 °C.

[0272] 7. Precipitate DNA at -20 °C for 30 min. Centrifuge at 12000 rpm and 4 °C for 10 min. At this time, a DNA precipitate appears at the bottom of the centrifuge tube.

[0273] 8. Discard the supernatant. Add 20 mL of 70% (v / v) ethanol solution. Gently pipette the precipitate with a pipette tip to make it float.

[0274] 9. Centrifuge at 12000 rpm for 8 min. Repeat the operation in step (8) once.

[0275] 10. Centrifuge at 12000 rpm for 8 min and discard the supernatant.

[0276] f. Dissolution of DNA

[0277] 11. Dry the precipitate at room temperature for 5 min to volatilize the residual ethanol. Add 0.5 mL of sterile water to dissolve the precipitate.

[0278] After grinding, the powder was directly added to the DNA extraction solution for extraction without sufficient incubation in the lysis buffer. Finally, only trace amounts of DNA were contained in the eluate. As detected by an ultraviolet spectrophotometer, the nucleic acid concentration was 5.30 μg / mL, which was significantly lower than the results of Example 1 and Example 2. The main purpose of incubating with the lysis buffer is to dissolve the cell membrane and nuclear membrane proteins using ionic surfactants such as (sodium dodecyl sulfate) SDS, rupture the cell membrane and nuclear membrane, and fully release DNA.

[0279] The present invention compared the processes and results of Example 1 and Comparative Example 1, as shown in Table 1.

[0280] Table 1. Comparison of the extraction processes and results between Example 1 and Comparative Example 1

[0281]

[0282]

[0283] In the above-mentioned examples and comparative examples, the agarose concentration was 1%, the buffer was 1×TAE buffer, the primers used for amplifying bacterial 16S rDNA were 27F and 1492R, and the primers used for amplifying fungal ITS were ITS1 and ITS4. The sequences of the two pairs of primers are as follows:

[0284] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0285] 1492R: 5'-GGTTACCTTGTTACGACTT-3'

[0286] ITS1: 5’-TCCGTAGGTGAACCTGCGG-3’

[0287] ITS4: 5’-TCCTCCGCTTATTGATATGC-3’

[0288] The PCR reaction system and reaction conditions are shown in Table 2:

[0289] Table 2. PCR reaction system and reaction conditions

[0290]

[0291] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0292] The above-described embodiments merely represent several implementation manners of the present invention, facilitating the specific and detailed understanding of the technical solution of the present invention. However, it should not be construed as a limitation to the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several deformations and improvements can still be made, and these all fall within the protection scope of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solution provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of this invention patent shall be subject to the content of the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A method for extracting total microbial DNA from moromi, characterized in that, the extraction method comprises the following steps: Mix a high-salt dilute-state soy sauce fermented moromi sample with a Tween 80 aqueous solution, centrifuge the resulting mixture, and collect the moromi precipitate; Under liquid nitrogen conditions, mix the moromi precipitate and the activated cation exchange resin and grind, and collect the powder; Mix the powder and the lysozyme buffer solution, incubate to prepare an incubation product; Mix the incubation product and the DNA extraction solution to extract DNA, centrifuge the resulting extraction product, and collect the supernatant; Mix the supernatant and ethanol for DNA precipitation, centrifuge the resulting precipitation product, collect the DNA precipitate, wash, and obtain the total microbial DNA.

2. The method for extracting total microbial DNA from moromi according to claim 1, characterized in that, the cation exchange resin meets the following conditions: (1) The cation exchange resin is a hydrogen-type cation exchange resin or a sodium-type cation exchange resin; (2) The steps of activation treatment include: soaking the cation exchange resin in an activator and removing the activator; (3) The dosage of the cation exchange resin is 10 wt%-50 wt% of the mass of the moromi sample; and, (4) The average particle diameter of the cation exchange resin is 4 nm-6 nm.

3. The method for extracting total microbial DNA from moromi according to claim 2, characterized in that, the steps of activation treatment have the following technical features: (1) The activator is an aqueous sodium chloride solution with a sodium chloride content of 5%-15% (w / v); (2) The soaking conditions include: the temperature is 15°C-30°C, and the time is 0.5 h-1.5 h; and, (3) The method for removing the activator includes centrifugation, the centrifugation speed is 8000 rpm-12000 rpm, and the centrifugation time is 4 min-6 min.

4. The method for extracting total microbial DNA from moromi according to claim 1, characterized in that, the Tween 80 aqueous solution meets the following conditions: (1) The content of Tween 80 in the Tween 80 aqueous solution is 0.2%-0.8% (w / v); and, (2) The dosage of the Tween 80 aqueous solution corresponding to each 1 g of the moromi sample is 1 mL-3 mL.

5. The method for extracting total microbial DNA from moromi according to any one of claims 1 to 4, characterized in that, the conditions for centrifuging the mixture include: the speed is 10000 rpm-14000 rpm, and the time is 10 min-20 min.

6. The method for extracting total microbial DNA from moromi according to any one of claims 1 to 4, characterized in that, the incubation meets the following conditions: (1) The dosage of the lysozyme buffer solution corresponding to each 3 g of the powder is 15 mL-25 mL; (2) The incubation temperature is 60°C-70°C, and the incubation time is 25 min-35 min.

7. The method for extracting total microbial DNA from moromi according to any one of claims 1 to 4, characterized in that, the extraction method meets the following conditions: The conditions for centrifuging the extraction product include: the rotation speed is 10,000 rpm - 14,000 rpm, and the time is 5 min - 15 min; and, The conditions for centrifuging the precipitation product include: the rotation speed is 10,000 rpm - 14,000 rpm, and the time is 5 min - 15 min.

8. The method for extracting total microbial DNA from moromi according to any one of claims 1 to 4, characterized in that, The cleaning agent used for cleaning the DNA precipitate is an aqueous ethanol solution with an ethanol volume concentration of 65% - 75%.

9. The method for extracting total microbial DNA from moromi according to any one of claims 1 to 4, characterized in that, The salt content of the moromi sample is 15 wt% - 25 wt%.

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