Method for extracting high-fidelity microbial flora from fermented grains

Abstract

This invention relates to the field of microbial technology, specifically to a high-fidelity method for extracting microbial communities from fermented mash, comprising the following steps: 1. Adding cell extraction solution 1 to the fermented mash sample, shaking, centrifuging, passing through a cell sieve, and collecting the supernatant into tube 1; 2. Adding cell extraction solution 2 to the remaining solid fermented mash, stirring, centrifuging, passing through a cell sieve, and collecting the supernatant into tube 1; 3. Centrifuging the supernatant in tube 1 to obtain bacterial precipitate 1, and resuspending bacterial precipitate 1 to obtain bacterial suspension 1; 4. Performing density gradient centrifugation on bacterial suspension 1 to obtain bacterial suspension 2, and centrifuging bacterial suspension 2 to obtain bacterial precipitate 2; 5. Washing bacterial precipitate 2, centrifuging, and collecting bacterial precipitate 3 as the fermented mash microbial community. The fermented mash cells extracted by the method of this application have advantages such as high recovery rate, speed, and support for Raman detection.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a high-fidelity method for extracting microbial communities from fermented grains. Background Technology

[0002] The quantity, composition, and metabolic activity of the microbial community, to a certain extent, determine the quality and taste of baijiu (Chinese liquor). To understand the essence of baijiu fermentation, one must start with the microorganisms. As a traditional open-air, solid-state, multi-strain fermented food, Maotai-flavor baijiu boasts a diverse and abundant microbial community. Furthermore, the fermentation process is influenced by the open micro-ecological environment, the dynamic changes in the local climate, and complex brewing techniques, making the rise and fall of the brewing microbial community extremely complex. Therefore, understanding the quantity and composition of the brewing microbial community is crucial for grasping the essence of Maotai-flavor baijiu brewing, ensuring stable quality, and improving the overall quality of the product.

[0003] Currently, amplicon and metagenomic high-throughput sequencing technologies are commonly used to analyze the composition and function of baijiu (Chinese liquor) microbial communities. However, these methods are time-consuming, costly, and cannot analyze the metabolic activity of microorganisms. Therefore, there is a need in this field for a method that can efficiently, accurately, quickly, and cost-effectively extract baijiu microbial community cells from complex baijiu mash samples. Summary of the Invention

[0004] To address the challenges of difficult microbial extraction, significant matrix interference, and low cell recovery rates in existing technologies for distillation mash samples, this invention provides a highly efficient, high-fidelity, high-throughput, and low-cost method for extracting microorganisms from distillation mash. This method offers advantages such as high cell recovery rate, rapid extraction, high purity, and support for Raman spectroscopy detection.

[0005] Specifically, this is achieved through the following technical solution: a method for high-fidelity extraction of microbial communities from fermented grains, comprising the following steps: 1. Add the mash sample to cell extraction solution 1, shake, centrifuge, pass through a cell sieve, and collect the supernatant into tube 1; 2. Add cell extract 2 to the remaining solid mash, stir, centrifuge, pass through a cell sieve, and collect the supernatant into tube 1; 3. Centrifuge the supernatant in tube 1 to obtain bacterial precipitate 1, and resuspend bacterial precipitate 1 to obtain bacterial suspension 1. IV. Perform density gradient centrifugation on bacterial suspension 1 to obtain bacterial suspension 2, and centrifuge bacterial suspension 2 to obtain bacterial precipitate 2. 5. Wash the bacterial precipitate 2, centrifuge, and collect the bacterial precipitate 3 as the fermentation mash microbial community.

[0006] In some specific embodiments, step one includes oscillation at room temperature; in some specific embodiments, the oscillation duration is 15 min to 25 min; in some specific embodiments, the oscillation includes oscillation at room temperature for 15 min to 25 min, for example, T can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min or a value within any two of these values.

[0007] In some specific embodiments, in step one, the centrifugation includes: centrifuging at a relative centrifugal force of 45×g to 55×g for 4 min to 6 min. In some specific embodiments, the centrifugation includes centrifugation at a relative centrifugal force of 45×g, 46×g, 47×g, 48×g, 49×g, 50×g, 51×g, 52×g, 53×g, 54×g, or 55×g for 4 min, 5 min, or 6 min. For example, the centrifugal force can be any value within the range of 45×g to 55×g, and the centrifugation time can be 4 min, 5 min, 6 min, or a value within the range of any two of these values.

[0008] In some specific embodiments, in step two, the cell extract 2 is a buffer solution containing a nonionic surfactant and a metal ion chelating agent.

[0009] In some specific embodiments, the nonionic surfactant is Tween 20.

[0010] In some specific embodiments, the metal ion chelating agent is sodium pyrophosphate.

[0011] In some specific embodiments, the protective agent is trehalose.

[0012] In some specific embodiments, the buffer solution is based on phosphate buffer.

[0013] In some specific embodiments, in step two, the cell extract 2 is prepared by the following method: using phosphate buffer as a matrix, Tween 20 is added to a volume ratio of 0.5%, sodium pyrophosphate is added to a final concentration of 25 mg, and trehalose is added to a final concentration of 0.5% to obtain a mixed solution. The pH of the mixed solution is then adjusted to 7.2 to obtain the cell extract.

[0014] In some embodiments, the stirring time in step two is 1.5 min to 2.5 min. In some specific embodiments, the stirring time in step two is 2 min.

[0015] In some embodiments, step two includes centrifuging at a relative centrifugal force of 45×g to 55×g for 4 to 6 minutes.

[0016] In some embodiments, in step three, the centrifugation speed is 8000 rpm to 12000 rpm and the centrifugation time is 4 min to 6 min; in some specific embodiments, in step three, the centrifugation speed is 10000 rpm and the centrifugation time is 5 min; in step three, the resuspension of bacterial precipitate 1 to obtain bacterial suspension 1 includes: resuspension of bacterial precipitate using PBS. In some specific embodiments, the ratio of the volume of PBS used for resuspension of bacterial precipitate to the fermented mash sample in step 1 is (1.5 to 2.5):1, expressed as ml:g; in some specific embodiments, the ratio of the volume of PBS used for resuspension of bacterial precipitate to the fermented mash sample in step 1 is 2:1.

[0017] In some specific embodiments, step four, the gradient centrifugation includes: slowly adding bacterial suspension 1 dropwise above iohexol solution, performing a first gradient centrifugation, and taking the bacterial suspension 2 obtained after the first gradient centrifugation for enrichment centrifugation to obtain bacterial precipitate 2.

[0018] In some embodiments, the first gradient centrifugation includes centrifugation at 12000×g to 16000×g for 35 min to 45 min.

[0019] In some embodiments, enrichment centrifugation includes centrifugation at 8000×g to 12000×g for 4 to 6 minutes. In some specific embodiments, enrichment centrifugation includes centrifugation at 10000×g for 5 minutes.

[0020] In some specific embodiments, the volume of the iohexol solution is equal to the volume of the bacterial suspension 1.

[0021] In some specific embodiments, the mass-volume concentration of the iohexol solution is 75% to 85%. In some specific embodiments, the mass-volume concentration of the iohexol solution includes 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%, for example, the concentration C can be 75%, 80%, 85%, or a value within the range of any two of these values.

[0022] In some specific embodiments, the iohexol solution is prepared by the following method: weigh 4g of iohexol, add 5ml of sterile water, and shake thoroughly to mix.

[0023] In some specific embodiments, in step one, the cell extract 1 is a phosphate buffer containing 0.5% Tween 20 by volume.

[0024] In some specific embodiments, in step one, the ratio of the fermented mash sample to the washing buffer is 1:(4~6) in g:ml. In some specific embodiments, the ratio is 1:4, 1:5, or 1:6, for example, the ratio can be 1:4, 1:5, 1:6, or a value within any range of these values.

[0025] In some specific embodiments, the amount of cell extract added in this application is the same as the amount of washing buffer added. To facilitate the detection of the extracted cells, Raman spectroscopy is generally performed on the extracted cells. The extraction method in this application reduces the use of cell extract, thereby avoiding any impact of the cell extract on cell viability and thus improving the viability of the extracted cells.

[0026] In some specific embodiments, in step one and / or, the pore size of the cell sieve is 35 μm to 45 μm; in some specific embodiments, the pore size of the cell sieve includes 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm or 45 μm or a value within the range of any two of these values.

[0027] In some embodiments, step five, the centrifugation includes centrifugation at 8000×g to 12000×g for 4 to 6 minutes. In some specific embodiments, step five, the centrifugation includes centrifugation at 10000×g for 5 minutes.

[0028] This invention employs a cell sieve method, resulting in fewer impurities in the obtained cell precipitate. Furthermore, this method is more convenient compared to other methods.

[0029] Beneficial Effects: Distilled grains are a complex system formed through fermentation using grains such as sorghum and rice husks as a substrate. Microorganisms not only adhere to the surface but also penetrate deep into the internal pores of the grains. This invention adds cell extract during the first extraction and employs a physical dispersion method combining high-frequency oscillation and stirring. This treatment effectively releases the microorganisms adhering to the surface and internal pores of the rice husks. Furthermore, trehalose is added to the cell extract; trehalose protects the released cells. The synergistic effect of these two methods results in a distilled grain microbial community with significantly high recovery rate and activity. The high-fidelity extraction method for microbial communities in distilled grains described in this application yields distilled grain cells with advantages such as high recovery rate, rapid extraction, and support for Raman spectroscopy detection. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other embodiments can be obtained based on these drawings.

[0031] Figure 1 This is a comparison diagram of cell viability after heavy water incubation of the recovered fermentation mash microbial community cells from the embodiments and comparative examples of this application; Figure 2 These are images obtained by microscopic imaging of the microbial community cells recovered from the embodiments and comparative examples of this application. Detailed Implementation

[0032] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0033] As mentioned in the background section, the microorganisms in fermented mash are diverse, including various types such as lactic acid bacteria, yeast, and mold. Their nutritional needs, growth environment preferences, and physiological characteristics vary greatly, which can easily lead to extraction distortion during the extraction process. Furthermore, as fermented mash is a highly acidic environment, the commonly used microbial extraction techniques are not suitable for extracting microbial communities from fermented mash. In order to solve the above problems, this application provides a method for high-fidelity extraction of microbial cells from fermented mash.

[0034] Instruments and reagents: PBS (0.01M, pH 7.2-7.4); 70 μm cell strainer; Tween 20; Iohexol; Sodium pyrophosphate (Aladdin, S489848); Stirrer; 1000 μL pipette tips; Multi-tube vortex mixer (Herto, MIX-200) DMT-2500; High-speed centrifuge (BECKMAN, ALLEGRA X-12).

[0035] This application discloses a method for high-fidelity extraction of microbial communities from fermented mash, comprising the following steps: 1. Add the mash sample to cell extraction solution 1, shake, centrifuge, pass through a cell sieve, and collect the supernatant into tube 1; 2. Add cell extract 2 to the remaining solid mash, stir, centrifuge, pass through a cell sieve, and collect the supernatant into tube 1; 3. Centrifuge the supernatant in tube 1 to obtain bacterial precipitate 1, and resuspend bacterial precipitate 1 to obtain bacterial suspension 1. IV. Perform density gradient centrifugation on bacterial suspension 1 to obtain bacterial suspension 2, and centrifuge bacterial suspension 2 to obtain bacterial precipitate 2. 5. Wash the bacterial precipitate 2, centrifuge, and collect the bacterial precipitate 3 as the fermented mash microbial community. In some specific embodiments, the shaking in step one includes shaking at room temperature; in some specific embodiments, the shaking duration is 15 min to 25 min; in some specific embodiments, the shaking includes shaking at room temperature for 15 min to 25 min, for example, T can be 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, or a value within any two of these values.

[0036] In some specific embodiments, in step one, the centrifugation includes: centrifuging at a relative centrifugal force of 45×g to 55×g for 4 min to 6 min. In some specific embodiments, the centrifugation includes centrifugation at a relative centrifugal force of 45×g, 46×g, 47×g, 48×g, 49×g, 50×g, 51×g, 52×g, 53×g, 54×g, or 55×g for 4 min, 5 min, or 6 min. For example, the centrifugal force can be any value within the range of 45×g to 55×g, and the centrifugation time can be 4 min, 5 min, 6 min, or a value within the range of any two of these values.

[0037] In some specific embodiments, in step two, the cell extract 2 is a buffer solution containing nonionic surfactants, metal ion chelating agents, and protective agents.

[0038] In some specific embodiments, the nonionic surfactant is Tween 20.

[0039] In some specific embodiments, the metal ion chelating agent is sodium pyrophosphate.

[0040] In some specific embodiments, the protective agent is trehalose.

[0041] In some specific embodiments, the buffer solution is based on phosphate buffer.

[0042] In some specific embodiments, in step two, the cell extract is prepared by the following method: using phosphate buffer as a matrix, Tween 20 is added to a volume ratio of 0.5%, sodium pyrophosphate is added to a final concentration of 25 mg, and trehalose is added to a final concentration of 0.5% to obtain a mixed solution. The pH of the mixed solution is then adjusted to 7.2 to obtain the cell extract.

[0043] In some embodiments, the stirring time in step two is 1.5 min to 2.5 min. In some specific embodiments, the stirring time in step two is 2 min.

[0044] In some embodiments, step two includes centrifuging at a relative centrifugal force of 45×g to 55×g for 4 to 6 minutes.

[0045] In some embodiments, in step three, the centrifugation speed is 8000 rpm to 12000 rpm and the centrifugation time is 4 min to 6 min; in some specific embodiments, in step three, the centrifugation speed is 10000 rpm and the centrifugation time is 5 min; in step three, the resuspension of bacterial precipitate 1 to obtain bacterial suspension 1 includes: resuspension of bacterial precipitate using PBS. In some specific embodiments, the ratio of the volume of PBS used for resuspension of bacterial precipitate to the fermented mash sample in step 1 is (1.5 to 2.5):1, expressed as ml:g; in some specific embodiments, the ratio of the volume of PBS used for resuspension of bacterial precipitate to the fermented mash sample in step 1 is 2:1.

[0046] In some specific embodiments, step four, the gradient centrifugation includes: slowly adding bacterial suspension 1 dropwise above iohexol solution, performing a first gradient centrifugation, and taking the bacterial suspension 2 obtained after the first gradient centrifugation for enrichment centrifugation to obtain bacterial precipitate 2.

[0047] In some embodiments, the first gradient centrifugation includes centrifugation at 12000×g to 16000×g for 35 min to 45 min.

[0048] In some embodiments, enrichment centrifugation includes centrifugation at 8000×g to 12000×g for 4 to 6 minutes. In some specific embodiments, enrichment centrifugation includes centrifugation at 10000×g for 5 minutes.

[0049] In some specific embodiments, the volume of the iohexol solution is equal to the volume of the bacterial suspension 1.

[0050] In some specific embodiments, the mass-volume concentration of the iohexol solution is 75% to 85%. In some specific embodiments, the mass-volume concentration of the iohexol solution includes 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85%, for example, the concentration C can be 75%, 80%, 85%, or a value within the range of any two of these values.

[0051] In some specific embodiments, the iohexol solution is prepared by the following method: weigh 4g of iohexol, add 5ml of sterile water, and shake thoroughly to mix.

[0052] In some specific embodiments, in step one, the cell extract 1 is a phosphate buffer containing 0.5% Tween 20 by volume.

[0053] In some specific embodiments, in step one, the ratio of the fermented mash sample to the washing buffer is 1:(4~6) in g:ml. In some specific embodiments, the ratio is 1:4, 1:5, or 1:6, for example, the ratio can be 1:4, 1:5, 1:6, or a value within any range of these values.

[0054] In some specific embodiments, the amount of cell extract 2 added in this application is the same as the amount of washing buffer added. To facilitate the detection of the extracted cells, Raman spectroscopy is generally performed on the extracted cells. The extraction method in this application reduces the use of cell extract, thereby avoiding any impact of the cell extract on cell viability and thus improving the viability of the extracted cells.

[0055] In some specific embodiments, in step one and / or, the pore size of the cell sieve is 35 μm to 45 μm; in some specific embodiments, the pore size of the cell sieve includes 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm or 45 μm or a value within the range of any two of these values.

[0056] In some embodiments, step five, the centrifugation includes centrifugation at 8000×g to 12000×g for 4 to 6 minutes. In some specific embodiments, step five, the centrifugation includes centrifugation at 10000×g for 5 minutes.

[0057] This invention employs a cell sieve method, resulting in fewer impurities in the obtained cell precipitate. Furthermore, this method is more convenient compared to other methods.

[0058] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below.

[0059] Example 1: A method for high-fidelity extraction of microbial communities from fermented mash 1) Weigh 3 g of sample, add 15 ml of cell extraction solution, and oscillate at high frequency for 20 min; 2) Centrifuge at 50×g for 5 min (4℃), and collect the supernatant through a 40 μm cell sieve into a 50 ml tube 1; 3) Add 15 ml of cell extract to the remaining solid mash and stir for 2 min; 4) Centrifuge at 50×g for 5 min (4℃), and collect the supernatant into tube 1 by passing it through a 40 μm cell sieve; 5) Centrifuge tube 1 at 10000×g for 5 min (4℃), discard the supernatant, and resuspend the bacterial pellet in 6 ml PBS; 6) Using a disposable sterile pipette, take 6 ml of the mash homogenate and slowly drop it onto an equal volume of 80% iohexol solution. Centrifuge at 14000×g for 40 min (4℃). 7) Take the cell layer, add an equal volume of sterile water and mix well. Centrifuge at 10000×g for 5 min (4℃) and collect the bacterial pellet.

[0060] 8) Discard the supernatant, take 1 ml of sterile ddH2O to wash the bacterial precipitate, and centrifuge at 10000×g for 5 min (4℃). 9) Discard the supernatant and store the collected bacterial precipitate in a 4°C refrigerator for later use.

[0061] The cell extract was prepared as follows: using PBS as the matrix, the final concentration of Tween 20 was 0.5%, the final concentration of trehalose was 0.5%, the final concentration of sodium pyrophosphate was 25 mM, and the pH was adjusted to 7.2. Preparation method of 80% iohexol solution: Weigh 4g of iohexol, add 5ml of sterile ddH2O, and shake thoroughly to mix.

[0062] Comparative Example 1 1) Weigh 2 g of sample and add it to 4 ml of 0.6% Tween 20 PBS, and shake at room temperature for 25 min; 2) Using a disposable sterile pipette, take 6 ml of the mash homogenate and slowly drop it onto an equal volume of 80% iohexol solution. Centrifuge at 14000 g for 60 min (4℃). 3) Transfer the cell layer above the iohexol solution into a 1.5 ml centrifuge tube and centrifuge at 10000 g for 5 min (4℃). 4) Discard the supernatant, take 1 ml of sterile ddH2O to wash the bacterial precipitate, and centrifuge at 10000 g for 5 min (4℃). 5) Discard the supernatant and store the collected bacterial precipitate in a 4°C refrigerator for later use.

[0063] Comparative Example 2 1) Weigh 1 g of sample into a special sampler, add 5 ml of PBS, and oscillate at high frequency for 5 min; 2) Take out the special sampler and centrifuge the homogenized slurry at low speed, 50 g for 1 min (4℃). 3) Take the supernatant and centrifuge at 10000 g for 5 min (4℃); 4) Discard the supernatant, take 1 ml of sterile ddH2O to wash the bacterial precipitate, and centrifuge at 10000 g for 5 min (4℃). 5) Discard the supernatant and store the collected bacterial precipitate in a 4°C refrigerator for later use.

[0064] Effect detection: Detection 1: Microscopic imaging was performed on the microbial community cells recovered from the fermented mash in Example 1, Comparative Example 1, and Comparative Example 2. The detection method is as follows: (1) Sample preparation: The bacterial suspensions obtained in Example 1, Comparative Example 1 and Comparative Example 2 were diluted, and 10 μL of each was added to a clean glass slide and covered with a coverslip to prepare the sample slide to be observed.

[0065] (2) Microscopic observation: Place the prepared sample slide on the stage of an optical microscope. Use a 40x objective lens for observation.

[0066] (3) Image acquisition: Images were randomly acquired from different fields of view for each sample slide. The focus of the microscope and the light intensity were adjusted to ensure that the cell morphology was clearly distinguishable.

[0067] (4) Result Analysis: The acquired images (such as...) Figure 2 The analysis was performed to compare the morphological integrity of cells and the presence of impurities in different samples.

[0068] Microscopic imaging results as follows Figure 2 As shown: by Figure 2 It can be seen that there are significant differences between Example 1 and the comparative example in terms of bacterial cell state and sample purity: Example 1: The obtained bacterial cell structure is intact, evenly distributed, and the background is clean with almost no interference from impurities, indicating that the method can efficiently and gently recover microorganisms. Comparative Example 1: Intact cells can also be recovered, and the background is clean with no interference from impurities. Comparative Example 2: A large number of non-specific impurities (such as obvious elongated and fine particles) are present in the field of view, and the cell morphology is severely damaged and the integrity is poor, indicating that the method causes significant damage to cells and introduces too many impurities during the recovery process. Therefore, only Example 1 and Comparative Example 1 will be analyzed in the following analysis.

[0069] Detection 2: The cell recovery rate of Example 1 and Comparative Example 1 was detected using a flow cytometer. The detection method is as follows: 1. Preparation of fluorescent cells: 1) Preparation method of fluorescent Escherichia coli: The tMuts strain was inoculated into 5 ml of LB medium containing ampicillin and cultured overnight at 37°C and 220 rpm. 1% was then transferred to a fresh 30 ml of LB medium containing ampicillin. When the OD... 600 When the concentration is 0.7, add 150 μL of IPTG, induce at 37°C for 4 h, and collect the bacterial cells.

[0070] 2) Preparation method of fluorescent yeast: Inoculate pyes2 into 5 ml SC-U medium and incubate overnight at 30℃ and 220 rpm. Take an appropriate amount of the overnight culture and add it to galactose induction medium to induce OD. 600 =0.3. The bacterial cells were collected after induction at 30℃ overnight for 18 hours.

[0071] 3) Divide the fluorescent coli and fluorescent yeast into n equal portions.

[0072] 2. The experimental steps for extracting microbial communities from fermented mash using fluorescent cells in Example 1 and Comparative Example 1 are as follows: Three parallel experimental groups were set up: Group 1 followed the method in Example 1, adding 1 ml of fluorescent bacterial culture to 3 g of sample; Group 2 followed the method in Example 1, adding 1 ml of fluorescent yeast culture to 3 g of sample; Group 3 followed the method in Example 1, adding only PBS without any fluorescent cells (as a background control). Comparative Example 1 was set up exactly the same as in Example 1. All groups underwent bacterial cell extraction according to their respective complete extraction procedures.

[0073] 3. Flow cytometer detection: The experimental procedures for the recovery rate of bacterial and fungal cells in Example 1 and Comparative Example 1 are as follows: 1) Indigenous microbial community of fermented mash: Microbial communities from fermented mash without added fluorescent strains were analyzed, and scatter plots of SSC-A and FSC-A were plotted. Due to their inherent size and particle size characteristics, the microbial community of fermented mash (mainly bacteria and a small amount of yeast) tends to concentrate in a specific region where the fluorescence signal is negative. This community is designated as phylum R1 (microbial community).

[0074] 2) Fluorescent Escherichia coli: Samples of fluorescent E. coli were processed, and a scatter plot of the FITC fluorescence channel and SSC-A was plotted. Due to their strong green fluorescence signal and relatively small cell size, fluorescent E. coli forms a unique population with high FITC and medium-low SSC levels. This population was designated as E (Escherichia coli).

[0075] 3) Fluorescent Yeast Phylum: Samples of fluorescent yeast were processed and a scatter plot of the DsRed fluorescence channel and SSC-A was plotted. Fluorescent yeast, due to its strong red fluorescence signal and significantly larger cell size and particle size, forms a unique population with high DsRed and high SSC / FSC ratios. This population was designated as Phylum Y (Yeast Phylum). This phylum should be completely distinct from Phylum R1, E, and P3. 4) Initial quantities of fluorescent E. coli and fluorescent yeast were determined separately: For *E. coli* fluoresceinii: resuspend one sample of *E. coli* cells in 1 ml of PBS, add 300 μL of counting microspheres, vortex to mix, and name the sample EC-0. Determine the cell concentration using flow cytometry absolute counting method; this concentration is the initial number of *E. coli* fluoresceinii. This experiment was performed in three independent replicates.

[0076] For fluorescent yeast: Resuspend one sample of fluorescent yeast cells in 1 ml of PBS, add 300 μL of counting microspheres, vortex to mix, and name the sample YC-0. Determine the cell concentration using flow cytometry absolute counting method; this concentration is the initial number of fluorescent yeast cells. This experiment was performed in three independent replicates.

[0077] 5) Measure the number of fluorescent cells recovered after treatment with different methods: For Example 1: 1 ml of PBS was used to resuspend the precipitate of fermented mash containing *E. coli* extracted using the method of Example 1, and 300 μl of counting microspheres was added. The mixture was then mixed and named Sample EC-1. Similarly, the precipitate of fermented yeast containing *E. coli* extracted using the method of Example 1 was resuspended, and counting microspheres were added. This sample was named Sample YC-1.

[0078] For Comparative Example 1: The same procedure was followed to prepare samples EC-2 (fluorescent Escherichia coli recovery sample of Comparative Example 1) and YC-2 (fluorescent yeast recovery sample of Comparative Example 1).

[0079] The concentration of fluorescent cells in samples EC-1, YC-1, EC-2, and YC-2 was determined using the absolute counting method by flow cytometry. Each sample was measured in triplicate.

[0080] 6) Calculation of absolute quantity: Based on the flow cytometry results, the absolute number of fluorescent Escherichia coli and fluorescent yeast before and after treatment in Example 1 and Comparative Example 1 was calculated using the absolute counting method.

[0081] Absolute counts are calculated using the following formula: .

[0082] In the formula, N cell : Absolute concentration of the fluorescent cell suspension to be tested; n cell : The number of events within the target cell gate (E gate or Y gate) collected by flow cytometry; n beads : The number of events within the microspheres collected by flow cytometry; V beads : Counting the volume of microspheres; V cell : Sample volume; c beads : Concentration of the original solution for counting microspheres.

[0083] 7) Cell recovery rate calculation: Cell recovery rate is calculated using the following formula: .

[0084] In the formula, N r : Number of fluorescent cells after pretreatment; N b : The original number of fluorescent cells.

[0085] 4. Detection Results: Cell recovery rate was determined using a flow cytometer. In Example 1, the treatment time was 87 min, the bacterial recovery rate was 86.91%, the fungal cell recovery rate was 98.28%, and the average recovery rate was 92.60%.

[0086] Comparative Example 1 was treated for 95 min, and the bacterial recovery rate was 82.34%, the fungal cell recovery rate was 94.09%, and the average recovery rate was 88.22%.

[0087] In summary, Example 1, while shortening the processing time, showed significantly higher bacterial and fungal recoveries and average recovery rates than Comparative Example 1, demonstrating that the method of the present invention has achieved significant progress in both recovery efficiency and quality. To further understand whether the method of Example 1 preserves the structural integrity of the original microbial community, the relative abundance of the microbial community recovered from the mash in Example 1 was subsequently tested.

[0088] Detection 3: Relative abundance was detected using 16S and 18S amplicon sequencing. The specific steps are as follows: 1) Weigh 3 g of sample and perform 16S and 18S amplicon sequencing respectively; 2) Weigh 3 g of sample and perform 16S and 18S amplicon sequencing after recovering the bacterial cells using Example 1; 3) Calculate the relative abundance based on OUT; ; ; ; 3. Test Results: The results of the sequencing relative abundance detection are shown in Table 1. As can be seen from Table 1, the relative abundance of bacteria recovered in Example 1 was 98.87%, the relative abundance of fungi recovered was 98.95%, and the total relative abundance was 98.90%.

[0089] Example 1 achieved a bacterial culture concentration exceeding 90% of the total bacterial community within 90 minutes (requiring coverage of species with a relative abundance >80% in the solid-state fermentation mash). Sequencing results showed that the relative abundance of key species in the recovered bacterial community of Example 1 was highly consistent with the original sample. This demonstrates the mild and non-selective nature of the method of this invention, avoiding damage or selective loss of specific microorganisms during extraction. Therefore, Example 1 provides a high-quality analytical sample that truly reflects the in-situ microbial ecology.

[0090] Table 1. Relative abundance detection results of six batches of fermented mash samples after pretreatment using the method in Example 1.

[0091] Test 4: Cell viability assay after heavy water incubation, specifically using the following method: 1) Obtain in situ microbial community cells of fermented mash by following the experimental steps of Example 1, Comparative Example 1, and Comparative Example 2 respectively; 2) The above-mentioned bacterial cells were resuspended in 50% D2O MRS medium and incubated for 0 h and 6 h, respectively; Background Raman spectra were acquired using Flow-RACS under the following conditions: laser output power of (60-200) mW, laser wavelength of 532 nm, exposure time of (1-3) s, and 60x objective lens, to subtract background signal interference. Raman spectra of single cells from samples of Example 1, Comparative Example 1, and Comparative Example 2 were acquired after incubation for 0 h and 6 h. One Raman spectrum was acquired from each cell, ensuring that the total number (n) of Raman spectra of the test samples after removing abnormal Raman spectra such as baseline unevenness and poor signal-to-noise ratio was greater than 200. 3) The acquired Raman spectral data were processed using R software to calculate the CDR (spectral value) of the sample; the detection results are as follows: Figure 1 As shown, it can be seen that the cell viability obtained by the method of this patent after incubation in heavy water is much higher than that of Comparative Example 3.

[0092] It is understood that the present invention has been described through some embodiments, and those skilled in the art will recognize that various changes or equivalent substitutions can be made to these features and embodiments without departing from the scope of the invention. Furthermore, under the teachings of the present invention, these features and embodiments can be modified to adapt to specific situations and materials without departing from the scope of the invention. Therefore, the present invention is not limited to the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of the present invention are protected by the present invention.

Claims

1. A method for high-fidelity extraction of microbial communities from fermented grains, characterized in that, Includes the following steps:

1. Add the mash sample to cell extraction solution 1, shake, centrifuge, pass through a cell sieve, and collect the supernatant into tube 1; 2. Add cell extract 2 to the remaining solid mash, stir, centrifuge, pass through a cell sieve, and collect the supernatant into tube 1; 3. Centrifuge the supernatant in tube 1 to obtain bacterial precipitate 1, and resuspend bacterial precipitate 1 to obtain bacterial suspension 1. IV. Perform density gradient centrifugation on bacterial suspension 1 to obtain bacterial suspension 2, and centrifuge bacterial suspension 2 to obtain bacterial precipitate 2.

5. Wash the bacterial precipitate 2, centrifuge, and collect the bacterial precipitate 3 as the fermentation mash microbial community.

2. The method as described in claim 1, characterized in that, In step two, the cell extract 2 is a buffer solution containing a nonionic surfactant, a metal ion chelating agent, and a protective agent; Preferably, the nonionic surfactant is Tween 20; Preferably, the metal ion chelating agent is sodium pyrophosphate; Preferably, the protective agent is trehalose; Preferably, the buffer solution is based on phosphate buffer.

3. The method as described in claim 1 or 2, characterized in that, In step two, the cell extract 2 is prepared by the following method: using phosphate buffer as a matrix, Tween 20 is added to a volume ratio of 0.5%, trehalose to a final concentration of 0.5%, and sodium pyrophosphate to a final concentration of 25mg to obtain a mixed solution. The pH of the mixed solution is then adjusted to 7.2 to obtain the cell extract.

4. The method as described in claim 1, characterized in that, In step four, the gradient centrifugation includes: slowly adding the bacterial suspension 1 dropwise above the iohexol solution, performing the first gradient centrifugation, and taking the bacterial suspension 2 obtained after the first gradient centrifugation for enrichment centrifugation to obtain bacterial precipitate 2.

5. The method as described in claim 4, characterized in that, The volume of the iohexol solution is equal to the volume of the bacterial suspension 1.

6. The method as described in claim 4, characterized in that, The mass-volume concentration of the iohexol solution is 75%–85%.

7. The method as described in claim 4, characterized in that, The iohexol solution was prepared by the following method: weigh 4g of iohexol, add 5ml of sterile water, and shake thoroughly to mix.

8. The method as described in claim 1, characterized in that, In step one, the cell extract 1 is a phosphate buffer containing 0.5% Tween 20 by volume.

9. The method as described in claim 1, characterized in that, In step one, the ratio of fermented mash sample to washing buffer is 1:(4~6), expressed in g:ml.

10. The method as described in claim 1, characterized in that, In step one and / or step two, the pore size of the cell sieve is 35μm~45μm.

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