Method for enriching dormant microbial cells from complex environment microbial community

Abstract

The invention discloses a method for enriching dormant microbial cells from complex environment microbial communities, and belongs to the field of microbial sample treatment and resource flora enrichment. According to the method, through a series of physical, chemical and enzymology synergistic steps, interference components such as impurities and DNA in a sample are effectively removed, anti-cracking dormant flora with metabolic potential is selectively enriched, the culturability and molecular detectability of the anti-cracking dormant flora are improved, and the method is suitable for subsequent function mining or metagenome analysis. The method can be operated in batches, and is an efficient and convenient enrichment method.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for enriching dormant microbial cells from complex environmental microbial communities, belonging to the field of microbial sample processing and resource microbial community enrichment. BACKGROUND

[0002] As a traditional fermentation agent, the potential active microbial composition of high-temperature Daqu directly affects the subsequent fermentation process, and directly affects the flavor, quality and stability of the terminal fermentation product. The preparation process of traditional high-temperature Daqu is divided into two stages: a fermentation stage of about 40 days, and a storage stage of 6 months. After the storage is over, the Daqu will be mixed with fresh wheat and used as mother Daqu, entering a new round of fermentation cycle. Therefore, it is crucial to study the potential active microbial community composition and its resuscitation potential in the seed Daqu for optimizing the application value of Daqu.

[0003] Dormancy (or "dormant state") refers to the low metabolic activity state of microorganisms under adverse environmental conditions (such as nutrient deficiency, extreme temperature, humidity change, etc.). However, dormant microbial communities are usually more resistant than microorganisms in active state, and can survive in extreme or adverse environments. In combination with the fermentation process of high-temperature Daqu, it can be known that the microbial community has undergone a transition from an initially nutrient-rich environment to a gradually unfavorable high-temperature and low-acid environment. In particular, the unique storage process of high-temperature Daqu forces the fermentation environment to gradually become dry (moisture content 12-15%) and nutrient-poor during the long-term storage of the Daqu block for up to 6 months. Therefore, under the stimulation of these environmental factors, the microbial community exhibits a significant decrease in metabolic rate but an increase in survival ability in order to maintain its own reproduction and metabolic activity, forming structures such as endospores (spores), chlamydospores or ordinary spores. These dormant bodies have strong resistance and environmental adaptability under natural conditions, and are the key basis for maintaining the fermentation activity of Daqu after long-term storage. In summary, understanding the dormancy rules of Daqu microbial community will help to optimize the storage environment and cycle, and avoid long-term dormancy of the microbial community and waste of storage resources caused by unsuitable conditions.

[0004] At present, with the strengthening of people's awareness of the importance of starter, more and more people begin to pay attention to the starter composition of high-temperature Daqu. Conventional amplicon sequencing combined with culturable strategy, as the gold standard for verifying the resuscitation potential of starter, has been widely used. However, limited by the principle of high-throughput sequencing technology, i.e. species identification based on indiscriminate DNA reading; especially in complex fermentation environment samples, the harsh fermentation conditions lead to the lysis and decline of microorganisms, so that active, dead, dormant bodies and extracellular DNA exist in the fermentation sample. Therefore, exploring the technical strategy of targeted screening of microorganisms with potential metabolic activity gradually attracts people's attention. Although the technical method based on marker recognition and pretreatment optimization has been reported in the identification of dormant bacterial flora, there are still limitations in complex samples, such as flow cytometry (limited resolution), Raman spectroscopy (low throughput), macro-transcriptome (low transcription volume) and membrane filtration (spore blockage). Therefore, there is an urgent need for a simple, efficient and suitable for complex sample dormant body enrichment strategy, which can accurately analyze the species composition and support the subsequent culturable verification. SUMMARY

[0005] The purpose of the present application is to provide an efficient pretreatment and enrichment method for dormant bacterial flora in complex fermentation environment samples, which can effectively remove impurities and DNA interference components in the sample through a series of physical, chemical and enzymatic coordination steps, selectively enrich anti-lysis dormant bacterial flora with metabolic potential, improve their culturability and molecular detectability, and be suitable for subsequent functional mining or metagenomic analysis.

[0006] The present application provides a pretreatment method for recovering dormant bacterial flora in complex fermentation environment samples, which comprises the following steps: (a) mixing the Daqu sample with sterile X1 solution, then shaking and centrifuging to obtain supernatant 1 and precipitate 1, wherein the sterile X1 solution is a sodium hexametaphosphate solution with a mass fraction of 0.5-1.0% prepared by PBS buffer, and the pH is adjusted to 7.0-7.2; (b) adding sterile X1 solution to the precipitate 1 obtained in step (a) and mixing uniformly, then homogenizing and centrifuging to obtain supernatant 2 and precipitate 2; (c) combining supernatant 1 and supernatant 2, centrifuging to obtain cell precipitate, then adding sterile X2 solution to the centrifuged precipitate and mixing thoroughly, and then treating with ultrasonic crushing to obtain cell suspension; The sterile X2 solution is: 5-50 mM sodium pyrophosphate, 0.1-1% Triton x100 is dissolved in a 0.70-0.85% mass fraction NaCl solution to obtain a mixed solution, and a compound lysozyme solution is added to the mixed solution in a ratio of (0.6-1.2):(80-120) by volume, wherein the compound lysozyme is prepared by mixing Yatalase 0.5-10 mg / ml, lysozyme 0.5-5.0 mg / ml, cellulase 2-20 mg / ml, yeast lyticase 2-20 μl, and proteinase K 1-10 μl; (d) centrifuging the broken cell suspension obtained in step (c) to obtain a cell precipitate, and washing the bacterial precipitate with PBS buffer; (e) heat lysis: adding TE buffer to the precipitate obtained in step (d) and placing it in a 50-85°C metal bath for 10 min, and then cooling; (f) enzymatic hydrolysis: adding lysozyme solution to the bacterial solution obtained after cooling in step (e) to make the final concentration of lysozyme 0.5-5.0 mg / ml, and incubating at 28-40°C and 150-300 rpm for 30-90 min to obtain an enzymatic hydrolysis bacterial solution; (g) membrane lysis: continuing to add NaOH and SDS solution to the enzymatic hydrolysis bacterial solution obtained in step (f), and then shaking and centrifuging to obtain a precipitate; (h) removing extracellular DNA: resuspending the precipitate obtained in step (g) with sterile water, shaking and mixing, and then centrifuging to obtain a bacterial precipitate; (i) enzymatic hydrolysis of residual DNA: adding PBS buffer to resuspend the bacterial precipitate obtained in step (h), adding DNase I enzyme buffer and DNA recombinant deoxyribonuclease, and treating at 25-40°C and 150-300 rpm for 10-40 min. Centrifuge the sample, discard the supernatant, and obtain a cell precipitate containing dormant bodies. Repeat the above washing steps at least once to obtain pure dormant body bacterial population.

[0007] In one embodiment of the present application, in step (a), the Daqu sample is mixed with the sterile X1 solution in a ratio of (0.5-1.0):(10-30) by mass volume, and shaken at 200-600 rpm for 30-90 min, and then centrifuged at 10-50 x g to obtain supernatant 1 and precipitate 1; In an embodiment of the present application, in step (b), the precipitate 1 obtained in step (a) is mixed with the sterile X1 solution at a mass / volume ratio of (0.5-1.0):(10-30), homogenized at 100-400 rpm for 1-3 min, and centrifuged at 10-50 x 103g for 1-5 min to obtain supernatant 2 and precipitate 2. g

[0008] In an embodiment of the present application, in step (c), the supernatant 1 and the supernatant 2 are combined, and centrifuged at 6000-12000 x 103g for 3-10 min to obtain a cell precipitate; the cell precipitate after centrifugation is mixed with the sterile X2 solution at a mass / volume ratio of (0.5-1):(2-10), and subjected to shock treatment at 150-300 rpm for 10-40 min, and then subjected to cell disruption under the condition of ultrasonic waves at 20-80 W to obtain a cell suspension. g

[0009] In an embodiment of the present application, in step (d), the cell suspension after disruption obtained in step (c) is centrifuged at 8000-12000 x 103g for 2-10 min to obtain a cell precipitate, and the cell precipitate is washed with PBS buffer. g

[0010] In an embodiment of the present application, in step (g), 1-5 mol / L NaOH solution and 2-10% SDS solution are added to the enzymatic bacterial solution obtained in step (f), and subjected to shock treatment at 150-300 rpm for 20-60 min; after the end, the precipitate is obtained by centrifugation at 6000-12000 x 103g for 2-5 min. g

[0011] In an embodiment of the present application, in step (h), the precipitate obtained in step (g) is resuspended with sterile water, and the bacterial concentration is 0.8-5.0 OD 600nm , and then mixed uniformly under the condition of 200-500 rpm, and the cell precipitate is obtained by centrifugation at 6000-12000 x 103g for 2-5 min. g In an embodiment of the present application, in step (i), the final concentration of DNase I enzyme is 3 U / μl-10 U / μl, and the final concentration of DNA recombinant deoxyribonuclease is 0.001-0.05 U / ml.

[0012] ​​​​​In an embodiment of the present application, the sterile X2 solution is prepared by dissolving 25 mM sodium pyrophosphate, 0.1% Triton x 100 in a 0.85% NaCl solution to obtain a mixed solution, and adding a complex lysozyme solution to the mixed solution at a ratio of 1:100 by volume, wherein the complex lysozyme is prepared by mixing Yatalase 5 mg / ml, lysozyme 2 mg / ml, cellulase 10 mg / ml, yeast lyticase 10 μl, and proteinase K 5 μl.

[0013] The present application provides a method for efficient enrichment of dormant bacterial population in complex fermentation samples, comprising the following steps: (a) Take a high-temperature Daqu sample, add X1 extraction solution, use a vortex shaker (Vortex) to adjust to 5 gears, shake for 40 min, and then centrifuge at 20 x g g for 1 min, collect the supernatant 1; (b) Continue to add 25 ml X1 extraction solution to the precipitate after (a) centrifugation, and mix well with a handheld blender (Blender) for 1-2 min, 20 x g g centrifuge for 1 min, collect supernatant 2; (c) Combine supernatant 1 and supernatant 2, and centrifuge at 8000 x g g for 5 min to obtain a cell precipitate (for subsequent processing of bacterial population); continue to add 5 ml X2 extraction solution to the precipitate after centrifugation and mix well to prepare a cell suspension, set 30 ℃, 200 rpm, and shake for 20 min; then use an ultrasonic wave (60W, break 2s, stop 3s) to break the instrument for 2 min; (d) Centrifuge the broken cell suspension at 12000 x g g for 2 min to obtain a cell precipitate, and discard the supernatant. Add 1 ml of 1 x PBS buffer to wash the bacterial precipitate, repeat the above operation 2 times to complete the washing of the lysed bacterial fragments. Finally, the obtained cell precipitate is placed in a 2 ml EP centrifuge tube for subsequent operation.

[0014] (e) Digest vegetative cells: add 900 μl of TE buffer to the 2 ml EP tube containing the precipitate, and perform the following 5-step operation according to the process of "heat lysis (1) - enzymolysis (2) - membrane lysis (3) - removal of extracellular DNA (4) - enzymolysis of residual DNA (5)"; (1) Heat lysis: place the bacterial solution obtained after step (e) in a 65 ℃ metal bath for 10 min; and wait for the temperature to drop to room temperature; (2) Enzymatic hydrolysis: Add 100 μl of 20 mg / ml lysozyme stock solution to the bacterial culture obtained in step (1) to achieve a final concentration of 2 mg / ml. Place the sample at 37 ℃ and 200 rpm for 30 min. (3) Membrane lysis: Add 250 μl of 3 mol / L NaOH and 250 μl of 6% SDS solution to the centrifuge tube from step (2), and shake at 200 rpm for 30 min at room temperature; after the process, centrifuge at 12000 xg for 5 min and discard the supernatant completely. At this point, most of the trophic cells have completed the digestion process, and the remaining precipitate contains only residual extracellular DNA and lysis-resistant dormant cells; (4) Removal of extracellular DNA: Using the dilution method, the viscous bacterial suspension obtained in step (3) was resuspended in 2 ml of sterile water. The bacterial cells were thoroughly vortexed and mixed for 2 min. After the bacterial cells were fully resuspended, the cell suspension was centrifuged at 12000 x g for 2 min, and the supernatant was discarded. The precipitate was washed three times to thoroughly remove residual DNA while avoiding the influence of residual reagents. (5) Enzymatic digestion of residual DNA: Subsequently, 450 μl of 1X PBS buffer, 50 μl of DNase I enzyme buffer, and 1 μl of recombinant DNase I were added to the bacterial pellet to resuspend the bacterial cells. The mixture was treated at 30 ℃ and 200 rpm for 20 min. The sample was then divided into 12000 × 10⁻⁶ samples. g After centrifugation for 2 min, the supernatant was discarded, yielding a cell pellet containing dormant cells. The pellet was resuspended in 5 ml of 1× PBS in a 10 ml centrifuge tube and centrifuged at 8000 x 1000 ml. g Centrifuge for 5 min to obtain cell pellet. Repeat the washing steps twice to obtain a pure dormant bacterial colony, which can be used for subsequent culture or gene sequencing. The specific models of the solution and the equipment used are as follows: The X1 extraction solution consisted of 1% sodium hexametaphosphate, 1× PBS buffer, and a pH of 7.2. The X2 extract consists of the following: The preparation method of the compound lysozyme is as follows: Prepare a stock solution by dissolving 5 mg / ml Yatalase, 2 mg / ml lysozyme, 10 mg / ml cellulase, 10 μl yeast lysozyme, and 5 μl proteinase K. Adjust the solution by adding 50 μl of the above compound enzyme solution to every 5 ml of X4 cell extract (25 mM sodium pyrophosphate, 0.1% Triton X100 dissolved in 0.85% NaCl solution, designated as X4).

[0015] The use amount of the high-temperature Daqu sample in step (a) and X1 buffer solution is: 1 g of Daqu added with 25 ml; The vortex instrument (Vortex-genie 2T, Scientific Industries) in step (a) is set to gear 5 and processed for 40 min; The added amount of the X1 solution in step (b) is 25 ml; the stirrer (RSD-180A, Rongshi) is set to medium gear and processed for 1-2 min; The added amount of the X2 solution in step (c) is 5 ml; the ultrasonic disrupter (SCIENTZ-IID, Xinzhibiological) is set to a crushing power of 60 W, and the crushing condition is that crushing is performed for 2 s and stopping is performed for 3 s; the total processing time is 2 min.

[0016] When the process in step (e) is performed to the third step, the intracellular contents are released due to the rupture of the cell membrane, so that the sample is instantaneously thick. The strategy of high-speed centrifugation combined with repeated washing is a very effective purification strategy when dealing with samples rich in “high-content dormant bodies”. Therefore, the present application needs to use the strategy of repeated washing (at least 3 times or more) with sterile water to eliminate the interference of extracellular impurities and DNA released by the system. At the same time, considering that there is still part of the residual DNA that has not been removed after processing, which will affect the subsequent DNA sequencing to some extent. Therefore, the present application uses Recombinant DNase I to completely digest the residual DNA, avoiding the result interference of the subsequent dormant body strategy results caused by the participation of DNA.

[0017] Beneficial effects 1) It can be operated in batches, and it is a kind of efficient and convenient enrichment method.

[0018] 2) The pretreatment process of the “high-content dormant body” sample is still effective.

[0019] 3) The sample after pretreatment can meet the sequencing and can also be cultivated. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 : The standard flow chart of extracting dormant body bacteria in a complex fermentation environment sample is explained.

[0021] Figure 2 : The influence of different cell extraction solutions on the extraction effect.

[0022] Figure 3 : The sample state of the vegetative cell after membrane lysis.

[0023] Figure 4 : The influence of different treatment strategies (H1-H5) on the cultivable fungal species and quantity.

[0024] Figure 5 Analysis of the structure difference of dormant body flora in the sample before and after the method of the present application. DETAILED DESCRIPTION

[0025] The information of the enzymes involved in the following examples is as follows: Yatalase purchased from: TaKaRa (Beijing, China), Cat. T017, enzyme activity: Chitinase ≥ 50 U / g, Chitobiase ≥ 500 U / g, Cell-wall lytic activity ≥ 10,000 U / g.

[0026] Lysozyme purchased from: Shanghai Generay Biotech, Cat. A610308-0005, enzyme activity ≥ 20000 U / mg.

[0027] Cellulase purchased from: Shanghai Generay Biotech, Cat. A002610-0001, enzyme activity ≥ 25 U / mg.

[0028] Yeast lyticase purchased from: Solabio (Beijing, China), Cat. R1020.

[0029] Proteinase K purchased from: TaKaRa (Beijing, China), Cat. 9034, enzyme activity ≥ 350 U / ml.

[0030] Recombinant DNase I enzyme purchased from: TaKaRa (Beijing, China), Cat. 2270A, enzyme activity - 5 U / μl.

[0031] Snailase purchased from: Solabio (Beijing, China), Cat. S8280-1g.

[0032] Reagent X1: sterile 1% sodium hexametaphosphate solution, solvent is 1 × PBS buffer; adjust pH to 7.2.

[0033] Preparation of complex lyticase solution: according to Yatalase 5 mg / ml, lysozyme 2 mg / ml, cellulase 10 mg / ml, yeast lyticase 10 μl, proteinase K 5 μl, configure into mother liquor.

[0034] X4 cell extract solution: 25 mM sodium pyrophosphate, 0.1% Triton ×100 dissolved in 0.85% NaCl solution.

[0035] Reagent X2: add 50 μl of the above complex lyticase solution to 5 ml of X4 cell extract solution.

[0036] Other reagents: sterile 1 x Tris-EDTA buffer (TE-buffer), sterile 20 mg / ml lysozyme solution, sterile 0.85% NaCl solution, 3 mol / L NaOH, 6% SDS solution, Recombinant DNase I (Takra, Cat. 2270A).

[0037] Relevant equipment: 0.22 μm micropore diameter 23 mm nitrocellulose filter head, 47 mm nitrocellulose filter membrane, shaking metal bath, sterile syringe, 50 ml sterile centrifuge tube, 10 ml sterile centrifuge tube, 2 ml sterile centrifuge tube, handheld stirrer, ultracentrifuge, vortex mixer, solid culture medium plate, incubator.

[0038] The high-temperature Daqu sample involved in the following examples is from a Maotai-flavor liquor producer in Renhuai District, Zunyi City, Guizhou Province.

[0039] Amplicon Sequencing involved in the following examples: a microbial community structure analysis method based on high-throughput sequencing technology. Its core principle is to perform PCR amplification on the target region (such as 16S rRNA gene, ITS region, etc.) by specific primers, and to perform deep sequencing on the amplification product, so as to obtain the classification composition information of microorganisms in the sample. Compared with the traditional culture method, Amplicon Sequencing has the advantages of high-throughput, high-sensitivity and rapid analysis of complex microbial community structure.

[0040] Example 1: Enrichment of dormant bodies in high-temperature Daqu samples The specific steps are as follows (the specific process is shown in Figure 1 , and Figure 1 the standard flowchart for extracting dormant bacterial flora in complex fermentation environment samples of the present application is explained in detail): 1. Preparation of solutions Sterile X1 solution: sterile 1% (w / w) sodium hexametaphosphate solution, solvent is 1 x PBS buffer; adjust pH to 7.2.

[0041] Preparation of recombinant lysozyme solution: according to Yatalase 5 mg / ml, lysozyme 2 mg / ml, cellulase 10 mg / ml, yeast lysozyme 10 μl, proteinase K 5 μl, configure into mother liquor.

[0042] X4 cell extraction solution: dissolve 25 mM sodium pyrophosphate and 0.1% Triton X100 in 0.85% NaCl solution.

[0043] Sterile X2 solution: Add 50 μl of the above-mentioned compound lysozyme solution to every 5 ml of X4 cell extract.

[0044] 2. Enrichment of dormant bodies in Daqu samples (a) Add 1 g of Daqu sample and 25 ml of sterile X1 solution to a 50 mL sterile test tube, mix thoroughly, and vortex at speed 5 for 40 min. After vortexing, centrifuge at low speed (under 20 × 10⁻⁶ conditions). g (1 min), carefully avoiding impurities, to obtain supernatant 1 and precipitate 1.

[0045] (b) Add 25 ml of sterile X1 solution to the precipitate 1 (per g) obtained in step (a) and mix well. Homogenize using a hand mixer at medium speed (1200 rpm) for 1 min to thoroughly disperse the matrix. Then, use a 20 × 10⁻⁶ ppm solution to homogenize. g Centrifuge for 1 min to obtain supernatant 2 and precipitate 2.

[0046] (c) Merge Supernatant 1 and Supernatant 2, using 8000 × g Centrifuge for 5 min to obtain cell pellet (for subsequent microbial treatment); add 5 ml of sterile X2 solution per g to the pellet and mix thoroughly; set the temperature to 30 °C, 200 rpm, and shake for 20 min; then treat with ultrasound (60 W, 2 s disruption, 3 s pause) for 2 min. (d) The fragmented cell suspension obtained in step (c) is subjected to a 12000 × [method / treatment]. g Centrifuge for 2 min to obtain cell pellet, discarding the supernatant. Add 1 ml of 1×PBS buffer to wash the bacterial pellet, repeating the above operation twice to wash away lysed bacterial fragments. Finally, transfer the obtained cell pellet to a 2 ml EP centrifuge tube for further processing.

[0047] (e) Digesting the nutrient body: Add 900 μl of TE buffer to the 2 ml EP tube containing the precipitate obtained in step (d); The operation is carried out according to the following procedure: "thermal lysis (1) - enzymatic digestion (2) - membrane lysis (3) - removal of extracellular DNA (4) - enzymatic digestion of residual DNA (5)"; the details are as follows: (1) Thermal lysis: The bacterial culture obtained after adding the buffer solution was placed on a 65 ℃ metal bath for 10 min; then cooled to room temperature. (2) Enzymatic lysis: 100 μl of 20 mg / ml lysozyme solution (purchased from Shanghai Biotechnology, item number: A610308-0005, enzyme activity ≥ 20000 U / mg) was added to the bacterial solution obtained from step (1) to make the final concentration of lysozyme 2 mg / ml, and incubated at 37 °C and 200 rpm for 30 min to obtain the enzyme lysed bacterial solution.

[0048] (3) Membrane lysis: 250 μl of 3 mol / L NaOH and 250 μl of 6% SDS solution were added to the enzyme lysed bacterial solution obtained from step (2), and treated at room temperature with 200 rpm for 30 min; after the end, 12000 × g centrifugation for 5 min, and the supernatant was completely discarded. At this point, most of the nutrient cells had completed the digestion process, and only residual extracellular DNA and anti-lysis dormant bodies remained in the remaining precipitate; (4) Removal of extracellular DNA: the viscous bacterial solution obtained from step (3) was resuspended with 2 ml of sterile water, and the bacterial cells were vortexed and mixed for 2 min. After the bacterial cells were fully suspended, the cell suspension was centrifuged at 12000 × g for 2 min, and the supernatant was discarded. The precipitate was washed for 3 times to fully wash away the residual DNA and avoid the influence of residual reagents; (5) Enzymatic lysis of residual DNA: then, 450 μl of 1 × PBS buffer solution was added to the bacterial cell precipitate to resuspend the bacterial cells, 50 μl of DNase I enzyme buffer solution, and 1 μl of DNA recombinant deoxyribonuclease (Recombinant DNase I) were added. The sample was treated at 30 °C and 200 rpm for 20 min. After centrifugation at 12000 × g for 2 min, the supernatant was discarded, and the cell precipitate containing dormant bodies was obtained. A 10 ml centrifuge tube was used, 5 ml of 1 × PBS was added to resuspend the precipitate, and the precipitate was centrifuged at 8000 × g for 5 min to obtain the cell precipitate. The above washing step was repeated for 2 times, and finally the pure dormant body bacterial population was obtained, which could be used for subsequent culture or gene sequencing.

[0049] (f) Culturable verification: then, 1 ml of sterile 1 × PBS buffer solution was added to the precipitate (dormant body bacterial population) obtained from step (e) to resuspend the precipitate to obtain a bacterial solution. The bacterial solution was adjusted to an appropriate concentration (3 × 10 6 cfu / ml), and 200 μl was taken to coat 10 solid LB and YPD plates respectively for growth verification.

[0050] (g) PCR verification: 30 typical morphological strains were picked from LB and YPD plates respectively for species identification, and a total of 10 mold and 10 bacterial species were identified at the species level, as shown in Table 1.

[0051] Comparative Example 1: The endospore bacterial population in high-temperature Daqu was extracted by the following method, which is described in the paper Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells.

[0052] Reagent X1: sterile 1% sodium hexametaphosphate solution, solvent: 1 × PBS buffer, adjust pH to 7.2.

[0053] Other reagents: sterile 1 × Tris-EDTA buffer (TE-buffer), sterile 20 mg / ml lysozyme solution, sterile 0.85% NaCl solution, 3 mol / L NaOH, 6% SDS solution, Recombinant DNase I (Takra, Cat. 2270A).

[0054] Related equipment: 0.22 μm micropore diameter 23 mm nitrocellulose filter head, 47 mm nitrocellulose filter membrane (NC), shaking metal bath, sterile syringe, 50 ml sterile centrifuge tube, 2 ml sterile centrifuge tube, homogenizer, ultracentrifuge, vortex mixer, solid culture plate, sterile suction flask, vacuum suction pump, incubator.

[0055] The high-temperature Daqu samples involved in the following examples were from a Jiangxiang Baijiu (liquor) producer in Renhuai District, Zunyi City, Guizhou Province.

[0056] The specific steps are as follows: 1. Use an ethanol flame sterilized metal spoon to add 3 g of high-temperature Daqu sample to a pre-weighed 50 ml sterile tube.

[0057] 2. Add 15 ml of 1% (w / w) sodium hexametaphosphate solution to the sterile test tube of step 1, and use a homogenizer at a speed of 17500 rpm for 1 min, stand for 2 min, and repeat the operation once to make it fully mixed; before use, sterilize the homogenizer with 70% ethanol or autoclave, and sterilize the homogenizer with 70% ethanol or autoclave.

[0058] 3. After step 2, let stand for 10 minutes. During this step, the heaviest particles will settle, while cells and any organic components will remain in solution. After standing, transfer the supernatant (containing microorganisms) to a clean 50 mL tube, being careful not to disturb the precipitate.

[0059] 4. Add 15 ml of sterile 1% sodium hexametaphosphate extract to the precipitate obtained in step 3 again, and then repeat steps 2 and 3. This repetition ensures maximum separation of grain particles from cells in the high-temperature koji. After carefully transferring the supernatant, use a 20× [method / method - likely a specific method or technique]. g Continue processing for 1 minute.

[0060] At this point, the precipitate is discarded, and the supernatant is combined. Subsequent steps will focus on further processing the supernatant.

[0061] 5. Using an autoclaved filtration flask, place a 47 mm nitrocellulose (NC) filter membrane into the filtration unit with sterile forceps. In a clean bench, divide the supernatant obtained in step 4 into two equal portions and add them to the filtration cup. One portion is used for culturing tests, and the other portion is stored at -80 °C for DNA sequencing.

[0062] First, the cells were collected onto an NC membrane using a vacuum pump. The NC membrane was then carefully handled with sterile forceps and placed in a sterile culture dish, stored at -80 °C for DNA sequencing.

[0063] Secondly, the filtrate was added to a new nitrocellulose (NC) filter membrane using a vacuum pump, and the NC membrane was picked up with sterile forceps and placed into a sterile culture dish. The filter membrane was then cut into small pieces with sterile scissors, and all membrane pieces were placed into sterile 2 ml centrifuge tubes for subsequent enrichment of endospores.

[0064] 6. Add 900 μl of 1× TE-buffer to a 2 ml centrifuge tube containing the second nitrocellulose membrane (NC) after step 5, and mix thoroughly using a vortex mixer. In this step, biomass will be transferred from the membrane into the TE buffer.

[0065] 7. Place the centrifuge tubes processed in step 6 into an incubator at 65 °C for 10 min, with a rotation speed of 500 rpm. After incubation, remove the centrifuge tubes from the incubator and allow them to cool for 15 min.

[0066] 8. Add 100 μl of freshly prepared lysozyme (purchased from Shanghai Sangon Biotech, catalog number: A610308-0005, enzyme activity ≥ 20000 U / mg) to the sample obtained in step 7, bringing the final concentration to 2 mg / ml. Incubate the sample at 37 °C for 60 min, maintaining a rotation speed of 500 rpm. These are the optimal conditions for lysozyme to lyse vegetative cells.

[0067] 9. After the lysis in step 8 is completed, add 250 μl of 3 mol / L sodium hydroxide (NaOH) and 250 μl of 6% sodium dodecyl sulfate (SDS) solution to the sample and let it stand at room temperature for 1 h (the paper mentions that the concentration of these surfactants is optimized so as not to damage the endospores and can effectively lyse the vegetative cells).

[0068] 10. After step 9, prepare a sterile filtration unit with a 25 mm diameter membrane. Place a sterile 0.22 μm NC membrane (25 mm diameter) inside. Add the sample processed in step 9 to the filtration unit and filter the liquid using a vacuum pump. Once the liquid has completely passed through, turn off the vacuum pump. At this point, lysed cellular material will not be retained on the membrane; only endospores will remain. Add 2 ml of sterile saline to the membrane to wash away any residual surfactant, and filter the liquid using the vacuum pump again. Once the liquid has completely filtered, turn off the vacuum pump. Leave the membrane in the filtration unit.

[0069] 11. Add recombinant DNase I enzyme to the membrane and perform DNase I treatment directly on the filter membrane. First, ensure the filter unit is leak-free and the vacuum pump is off. If possible, perform this digestion in a room slightly above room temperature, as the enzyme works best at temperatures above 25 °C. Note: Placing an alcohol lamp next to the filter unit can increase the temperature and help maintain a sterile environment, thus reducing the risk of sample contamination.

[0070] After recombinant DNase I digestion is complete, turn on the vacuum pump to remove the enzyme from the sample. Add 1 ml of physiological saline to the filter unit and filter to wash away any residual enzyme. Once the liquid has completely passed through, turn off the vacuum pump. Use sterile forceps to remove the filter membrane containing endospores and add 1× PBS buffer to elute the bacteria on the NC membrane.

[0071] 12. Adjust the bacterial culture to a suitable concentration and spread it onto 10 solid LB and YPD plates respectively for growth verification.

[0072] 13. PCR verification: Thirty typical morphological strains were selected from LB and YPD plates for species identification. A total of 10 fungi and 10 bacterial microorganisms were identified at the species level, as detailed in Table 1.

[0073] Note: When processing complex samples containing a large number of Bacillus subtilis and their endospores, the traditional NaOH and SDS membrane lysis steps often lead to a significant viscous phase in the system. This is because the polysaccharides and other high-molecular-weight substances released after bacterial cell rupture have strong hydration properties, causing the system to exhibit a thick, gel-like consistency (see...). Figure 3 This can lead to clogging of subsequent membrane filtration steps, a significant reduction in efficiency, and even make it difficult to continue operation. Figure 3 The image shows the sample state after vegetative cells are lysed via membrane. The figure illustrates that after membrane lysis (NaOH + SDS) treatment, the sample exhibits a distinctly gel-like, viscous state. The right image shows the droplet state of the same sample, demonstrating extremely high viscosity. This phenomenon may be due to the release of polysaccharides, proteins, and other macromolecules during the lysis process, leading to enhanced solution hydration. This characteristic suggests that in complex samples rich in Bacillus or endospores, a strategy combining dilution and high-speed centrifugation should be used instead of membrane filtration to avoid sample clogging and processing failure.

[0074] To address this issue, this invention systematically optimizes the pretreatment steps for cell precipitation, release, and enrichment. Furthermore, it introduces a dilution + high-speed centrifugation strategy based on DNA water solubility after the membrane lysis step. This effectively reduces system viscosity, removes extracellular DNA, and achieves efficient separation of dormant cells from residual DNA, fundamentally overcoming the limitations of traditional methods in high-spore, high-impurity samples, which suffer from poor universality and susceptibility to failure. Through these improvements, this invention significantly enhances the operability and stability of the sample processing procedure. To further verify the advantages of this method, this paper systematically compares the differences between this invention (Example 1) and the traditional method (Comparative Example 1) in terms of the types of culturable dormant cells. The results are shown in Figure 1.

[0075] Advantages of the present invention: "Example 1" has four major advantages over "Comparative Example 1". (1) By adopting a strategy of combining gentle vortex oscillation with hand stirring in the early stage, the microbial community in the complex fermentation sample is fully released, which significantly reduces the damage to the cell structure that may be caused by simple high-speed homogenization, thereby improving the integrity and survival rate of dormant cells. (2) The present method introduces a synergistic strategy of compound lysozyme combined with short-range ultrasonic disruption, which shortens the operation time by half (from 60 min to 30 min) compared with the traditional long-term "lysozyme and membrane lysis" treatment, while improving the lysis efficiency of nutrient cells and significantly optimizing the effect of subsequent membrane lysis and DNA removal steps. (3) In response to the common problem of "sample viscosity and filter membrane blockage after release of intracellular substances" in the treatment process, the present invention introduces the idea of ​​aqueous dilution + high-speed centrifugation to replace membrane filtration, which effectively breaks the dependence of traditional methods on membrane filtration, making the method more adaptable and universal. (4) The results of culturable verification show that this method is superior to Comparative Example 1 in terms of the recovery of culturable dormant species and low abundance species. In particular, it shows a stronger enrichment capacity in terms of bacterial and fungal diversity, and has higher potential for microbial resource mining and application expansion.

[0076] Example 2: Using the same procedure as in Example 1, the concentration of the extraction reagent and parameters were optimized. Reagent parameters: Lysozyme and cellulase were purchased from Sangon Biotech (product numbers: A610308-0005, A002610-0001), Yatalase was purchased from TaKaRa (Cat. T017), yeast lysozyme was purchased from Solarbio (Cat. R1020), and proteinase K was purchased from TaKaRa (Cat. 39450-01-6).

[0077] 1. The effect of the formulation of the main cell extract (sterile X1 solution) on the extraction effect of the microbial community. The operation process of this embodiment is basically the same as that of Example 1, except that the components of the cell extract solution are adjusted: the sterile X1 solution is replaced with a 25 mM sodium pyrophosphate solution containing 0.5% Tween 20 (hereinafter referred to as X3) to evaluate the effect of surfactant-assisted extraction on cell release and dormant body enrichment.

[0078] Experimental results are as follows Figure 2 As shown ( Figure 2 The effect of different cell extraction solutions on extraction efficiency. a. Physical state of samples after treatment with X1 cell extraction solution (1% sodium hexametaphosphate); b. Physical state of samples after treatment with X3 cell extraction solution (25 mM sodium pyrophosphate + 0.5% Tween 20); c. Results of differences in bacterial community structure obtained by high-throughput sequencing under different extraction solution treatments. Figure 2 Figure ab shows the differences in the physical state of the two extracts, X1 and X3, during sample processing. The results indicate that X3, due to the presence of Tween 20, exhibits strong foaming properties, generating a large amount of foam during shaking, which significantly affects the dispersion efficiency and homogenization of the sample, and easily leads to solution loss. In contrast, extract X1 is more stable, less prone to foaming, and more suitable for continuous processing of complex samples.

[0079] Figure 2 c. The results showed that X1 (1% sodium metaphosphate) was more effective in extracting dormant bacterial flora. Amplicon sequencing results showed that samples treated with X1 were significantly enriched at the phylum Firmicutes (…). Bacillota ) and Actinobacteria ( Actinomycetota The presence of typical anti-lysed dormant bacterial groups, such as X1 extract, indicates that X1 extract is more conducive to the release and subsequent detection of dormant bacterial groups in samples from complex environments.

[0080] In summary, although X3 has a certain cell surface cleaning effect, it has strong foaming properties, unstable operation and limited enrichment effect. Therefore, this invention prefers X1 extract as the standard cell release solution to ensure the stability of subsequent steps and the accuracy of dormant cell enrichment.

[0081] 2. Effects of different enzyme pretreatments (sterile X2 solution) on culturability and microbial community structure (1) The specific implementation method is the same as in Example 1, except that the composition of the exogenous enzyme in the sterile X2 solution is optimized, as follows: 1) Control group H1: Phosphate-buffered saline (PBS); The specific implementation method is the same as in Example 1, except that the sterile X2 solution in step (c) is changed to phosphate buffered saline (PBS), and the temperature is set at 30 °C, 200 rpm, and shaken for 20 min; the sample is processed according to the method in Example 1. 2) Control group H2:X4 cell extract; The specific implementation method is the same as in Example 1, except that the sterile X2 solution in step (c) is changed to X4 cell extract (25 mM sodium pyrophosphate, 0.1% Triton X100, dissolved in 0.85% NaCl solution), and the temperature is set at 30 ℃, 200 rpm, and shaken for 20 min; the sample is processed according to the method in Example 1. 3) Experimental group H3: Cell extract with 30 mg / L snail enzyme added X4; The specific implementation method is the same as in Example 1, except that the sterile X2 solution in step (c) is adjusted to cell extract X4 with 30 mg / L snailase added (25 mM sodium pyrophosphate, 0.1% Triton X100, and 30 mg / L snailase are dissolved in 0.85% NaCl solution, respectively), and the temperature is set at 30 °C, 200 rpm, and shaken for 20 min; the sample is processed according to the method in Example 1. 4) Experimental group H4: Cell extract X4 supplemented with 100 mg / L cellulase: The specific implementation method is the same as in Example 1, except that the sterile X2 solution in step (c) is adjusted to cell extract X4 with 100 mg / L cellulase added (25 mM sodium pyrophosphate, 0.1% Triton ×100, and 100 mg / L cellulase are dissolved in 0.85% NaCl solution), and the temperature is set at 30 °C, 200 rpm, and shaken for 20 min; the sample is processed according to the method in Example 1. 5) Experimental group H5 (Example 1): Cell extract with added compound enzyme X4: The specific implementation method is the same as in Example 1, and the sample is processed according to the method in Example 1; (2) In order to objectively evaluate the impact of different pretreatment strategies on the culturability of dormant microbial communities, this invention systematically compared Daqu samples treated with different methods (numbered H1-H5).

[0082] 1) Microscopic observation and preliminary characterization: First, the morphology of the bacterial culture in each treatment group was observed using an optical microscope.

[0083] The results showed that the cell morphology of the cells did not change significantly after treatment with different enzyme solutions, indicating that it is difficult to determine the effect of the treatment strategy on the release of dormant cells based solely on microscopic observations. Therefore, further verification experiments were conducted from the perspective of culturable species.

[0084] 2) Analysis of culturable microbial communities (taking fungi as an example): First, using microscopy, a rough estimate of the bacterial concentration for culture was made, and it was adjusted to (3 × 10⁻⁶). 6 Take 200 μL of dormant bacterial cultures obtained by different treatment methods (cfu / ml), and administer them separately at 10... -1 10 -2 10 -3 and 10 -4Concentration gradients were applied to plates corresponding to three different culture media (PDA, YPD, and Czapek's high-salt medium). Two plates were prepared for each concentration gradient on each medium, for a total of 24 plates per group (4 gradients × 3 media × 2 replicates). All plates were cultured under identical conditions. After cultivation, based on colony morphology, 30 representative strains were selected from each group (H1-H5) for purification, classification, and statistical analysis. A total of 150 culturable strains were obtained (5 groups × 30 strains) to evaluate the impact of different treatments on bacterial diversity and culturability.

[0085] like Figure 4 As shown, Figure 4 The effects of different treatment strategies (H1-H5) on the species and quantity of culturable fungi were investigated. H1: Phosphate-buffered saline (PBS); H2: Cell extract (X4); H3: 30 mg / L snailase dissolved in X4 buffer; H4: 100 mg / L cellulase dissolved in X4 buffer; H5: 50 μl of a compound enzyme solution (Yatalase - 5 mg / ml, lysozyme - 2 mg / ml, cellulase - 10 mg / ml, yeast lysozyme - 10 μl, proteinase K - 5 μl) dissolved in 5 ml of X4 buffer. The results showed that the number of culturable dormant fungi was significantly increased in the H5 treatment group. Colors represent different fungal taxa, and a total of 25 fungal species were identified. The line graph represents the number of filamentous fungi in each treatment group.

[0086] The results showed that PBS buffering treatment did not effectively release dormant bacterial communities, resulting in low abundance and diversity of species.

[0087] In contrast, the series containing X2 buffer (H2-H4) showed a steady increase in both species diversity and abundance range, which may be closely related to the increased spore species released after complete cell lysis.

[0088] Ultimately, the treatment with the compound enzyme (the method in Example 1) yielded the best culturable results, which fully demonstrates that complete cell lysis has a positive promoting effect on spore release and can significantly increase the culturable proportion of the sample.

[0089] Example 3: Comparison of microbial community structure 1. Specifically, as in Example 1, evaluate the differences in bacterial community structure between the untreated group (original Daqu sample) and the present invention (dormant bacterial community obtained using the method of Example 1). Specifically, the present invention compares the amplicon sequencing results of the pretreated bacterial community structure.

[0090] In this study, for bacterial communities, the V3-V4 hypervariable region was selected for 16S rRNA gene amplification; for fungal communities, the ITS1 region was targeted for sequence amplification. Bioinformatics processing, including filtering, splicing, dechimericating, clustering, and species annotation, was performed on the amplicon sequences to obtain the relative abundance and distribution characteristics of each community in the samples. This was used to assess the changes in microbial structure in the original Daqu samples before enrichment and in the dormant bacterial communities obtained after enrichment.

[0091] like Figure 5 As shown, Figure 5 This figure illustrates the structural differences in dormant bacterial communities in samples before and after treatment using the method of this invention. It shows the relative abundance changes at the genus level of detectable fungi (top) and bacteria (bottom) in samples treated with the method of this invention, compared to samples tested using the conventional method (direct sample, untreated group).

[0092] The results showed that the bacterial community after pretreatment and enrichment underwent significant changes in population composition, with bacteria mainly consisting of... Kroppenstedtia, Virgibacillus, Bacillus, Oceanobacillus Firmicutes ( Bacillota The main members are members; the fungal community is mainly composed of... Thermoascus, Wallemia Highly tolerant groups were significantly enriched. These dominant strains generally possess the ability to form spores, chlamydospores, or dormant spores, and are typical lysis-resistant and stress-tolerant microorganisms, representing the core components of dormant bodies in fermentation systems.

[0093] Compared to the untreated original sample, the relative abundance of lysis-resistant bacteria in the microbial community enriched using this method was significantly increased, indicating that the strategy of this invention has a significant selective enrichment effect on dormant organisms in complex samples. Simultaneously, this method effectively overcomes the limitations of traditional sequencing technologies, accurately assessing the relative abundance of dormant organisms in the fermentation system. By combining it with metagenomic sequencing, the functional potential of these dormant organisms can be further explored, enabling subsequent studies to more comprehensively understand the types and composition of dormant organisms in the fermentation environment and achieve multi-dimensional integrated research from both cultivar and functional perspectives.

[0094] 2. Following the same procedures as Example 1 and Comparative Example 1, after the experiments, the culturability of the obtained dormant organisms was tested using culturability verification experiments (specifically, the culturability verification of Example 1 or the PCR verification of Comparative Example 1). The results are shown in Table 1. Table 1: Differences in the effectiveness of Example 1 and Comparative Example 1 in terms of the types of culturable dormant organisms

[0095] Nd: not detected; used to indicate that the culture was unsuccessful.

[0096] Example 4: Result Comparison The specific methods are the same as in Examples 1 and 2, and Comparative Example 1. The advantages and disadvantages of the three methods in Example 1, Comparative Example 1 and Example 2 are compared, and the results are shown in Table 2.

[0097] Table 2: Comprehensive Comparison of the Three Methods

[0098] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A pretreatment method for recovering dormant bacterial communities from samples of complex fermentation environments, characterized in that, The method includes the following steps: (a) After mixing the Daqu sample with sterile X1 solution, shake and centrifuge to obtain supernatant 1 and precipitate 1. The sterile X1 solution is a sodium hexametaphosphate solution with a mass fraction of 0.5~1.0% prepared with PBS buffer and adjusted to pH 7.0~7.

2. (b) Add sterile X1 solution to precipitate 1 obtained in step (a), mix well, homogenize, centrifuge and obtain supernatant 2 and precipitate 2; (c) Combine supernatant 1 and supernatant 2, centrifuge to obtain cell pellet; add sterile X2 solution to the pellet after centrifugation, mix thoroughly, shake, and then sonicate to obtain cell suspension. The sterile X2 solution is prepared by dissolving 5-50 mM sodium pyrophosphate and 0.1-1% Triton ×100 in a 0.70-0.85% NaCl solution to obtain a mixed solution. A compound lysozyme solution is then added to the mixed solution at a volume ratio of (0.6-1.2):(80-120). The compound lysozyme is prepared by mixing 0.5-10 mg / ml Yatalase, 0.5-5.0 mg / ml lysozyme, 2-20 mg / ml cellulase, 2-20 μl yeast lysozyme, and 1-10 μl proteinase. (d) Centrifuge the broken cell suspension obtained in step (c) to obtain cell pellet, and wash the bacterial pellet with PBS buffer; (e) Thermal pyrolysis: Add TE buffer to the precipitate obtained in step (d) and place it in a metal bath at 50~85℃ for 10 min, then cool it down. (f) Enzymatic hydrolysis: Add lysozyme solution to the bacterial solution obtained in step (e) after cooling, so that the final concentration of lysozyme reaches 0.5~5.0 mg / ml, and incubate at 28~40 °C and 150~300 rpm for 30~90 min to obtain the enzymatically hydrolyzed bacterial solution; (g) Membrane lysis: Add NaOH and SDS solution to the enzymatic hydrolysate obtained in step (f), shake and centrifuge to obtain a precipitate; (h) Removal of extracellular DNA: The precipitate obtained in step (g) is resuspended in sterile water, shaken and mixed, and then centrifuged to obtain a bacterial precipitate; (i) Enzymatic digestion of residual DNA: Add PBS buffer to the bacterial pellet obtained in step (h) to resuspend the bacterial cells, add DNase I enzyme buffer and DNA recombinant deoxyribonuclease, and treat at 25-40 °C and 150-300 rpm for 10-40 min; after centrifuging the sample, discard the supernatant to obtain a cell pellet containing dormant cells, repeat the above washing steps at least once to finally obtain a pure dormant bacterial colony.

2. The method according to claim 1, characterized in that, In step (a), the Daqu sample and sterile X1 solution are mixed at a material-to-liquid ratio of (0.5~1.0):(10~30), and shaken at 200~600 rpm for 30~90 min. g After centrifugation for 1-5 minutes under the specified conditions, supernatant 1 and precipitate 1 were obtained.

3. The method according to claim 1 or 2, characterized in that, In step (b), the precipitate 1 obtained in step (a) is mixed with sterile X1 solution at a material-to-liquid ratio of (0.5~1.0):(10~30), and homogenized at 500~1200 rpm for 1~3 min, followed by 10~50× g After centrifugation for 1-5 minutes under the specified conditions, supernatant 2 and precipitate 2 were obtained.

4. The method according to any one of claims 1 to 3, characterized in that, In step (c), supernatant 1 and supernatant 2 are merged, 6000~12000× g After centrifugation for 3-10 min under the specified conditions, cell pellet was obtained. The pellet was mixed with sterile X2 solution at a mass-to-volume ratio of (0.5-1):(2-10). The mixture was shaken at 150-300 rpm for 10-40 min, and then the cells were disrupted by sonication at 20W-80W for 2-5 min to obtain a cell suspension.

5. The method according to any one of claims 1 to 4, characterized in that, In step (d), the broken cell suspension obtained in step (c) is heated to 8000~12000× g After centrifugation for 2-10 min under the specified conditions, cell pellet was obtained and washed with PBS buffer.

6. The method according to any one of claims 1 to 5, characterized in that, In step (g), add 1-5 mol / L NaOH solution and 2-10% SDS solution to the enzymatically hydrolyzed bacterial culture obtained in step (f), and shake at 150-300 rpm for 20-60 min; after the process, shake at 6000-12000× g Centrifuge for 2-5 minutes to obtain a precipitate.

7. The method according to any one of claims 1 to 6, characterized in that, In step (h), the precipitate obtained in step (g) is resuspended in sterile water, and the bacterial concentration is 0.8~5.0 OD. 600nm After mixing by shaking at 200-500 rpm for 2-5 min, then at 6000-12000 × g Centrifuge for 2-5 minutes under the specified conditions to obtain bacterial precipitate.

8. The method according to any one of claims 1 to 6, characterized in that, In step (i), the final concentration of DNase I enzyme added is 3 U / μl to 10 U / μl, and the final concentration of recombinant DNA deoxyribonuclease is 0.001 to 0.05 U / ml.

9. The method according to any one of claims 1 to 6, characterized in that, The sterile X2 solution is prepared by dissolving 25 mM sodium pyrophosphate and 0.1% Triton ×100 in 0.85% NaCl solution to obtain a mixed solution. A compound lysozyme solution is then added to the mixed solution at a volume ratio of 1:

100. The compound lysozyme is prepared by mixing 5 mg / ml Yatalase, 2 mg / ml lysozyme, 10 mg / ml cellulase, 10 μl yeast lysozyme, and 5 μl proteinase K.

10. The dormant bacterial flora is recovered by any one of the methods described in claims 1 to 9.

Images