Efficient and low-loss single cell extraction method in complex flora and application in vegetative body-dormant body separation and living cell detection

Abstract

The invention discloses an efficient and low-loss single cell extraction method in complex flora and application in vegetative mass-dormant mass separation and living cell detection, and belongs to the technical field of microbial ecology. According to the method, an efficient and low-loss Nycodenz-Percoll extraction strategy is developed, the problems of low extraction efficiency, flora extraction structure deviation and the like of a traditional method are solved, and the method can effectively distinguish vegetative bodies from dormant bodies. According to the method, the microbial flora can be efficiently and nondestructively extracted from a complex matrix, and live / dead staining reagents are integrated to identify the physiological status. Meanwhile, according to the method, Percoll density gradient centrifugation is further fused for testing, and secondary separation can be further carried out on part of dormant bodies and vegetative cells, so that the purpose of efficient and graded purification is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to a high-efficiency and low-loss single cell extraction method in a complex bacterial community and its application in vegetative-sporal separation and live cell detection, belonging to the technical field of microbial ecology. BACKGROUND

[0002] As a small-scale model of human understanding of natural environment, the core participants and functional executors of micro-ecological network are very important for understanding ecological processes. Among them, active microorganisms as functional executors in complex bacterial communities, capturing and analyzing these metabolically active microorganisms are the key to understanding and remodeling ecological bacterial communities. With the development of traditional sequencing technology, high-throughput sequencing technology based on indiscriminate inclusion of genetic material enables people to understand the structure and function of complex bacterial communities from a global perspective. However, in its essence, metagenomics technology focuses more on gene-level transmission and expression, and lacks recognition of bacterial community metabolic activity phenotype and physiological state, which limits its application in active microorganism resource mining. Therefore, the establishment of a technology system for identifying active microorganism bacterial communities has gradually attracted people's attention.

[0003] RNA extraction is often considered an important prerequisite for identifying active microorganisms in bacterial communities. However, complex environmental bacterial communities are often affected by high temperature, low water activity, high salt concentration, and complex physicochemical factors such as natural pigments. Microbial cells in the sample are often in a stressed state, intracellular RNA is prone to degradation, and the overall RNA extraction amount is low. This makes it a very challenging task to extract sufficient and high-quality RNA from such extreme or special environments, especially when subsequent macro-transcriptome or RNA-based metagenome sequencing is required, which requires higher requirements for RNA integrity and representativeness. Traditional RNA extraction methods often face low extraction efficiency, many residual inhibitors, and serious downstream amplification interference in such environmental samples, which seriously restricts the accurate analysis of the functional state of microorganisms in complex communities. For example: although the macro-transcriptome strategy has been widely concerned in the active recognition layer, the development of 16S-RNA-seq technology has been proven to be unable to be applied to complex communities. Therefore, the current technical system urgently needs to develop high-quality single cell extraction technology as the basis for effective separation and identification of functional microorganisms.

[0004] Currently, single-cell RNA sequencing, droplet microfluidics, FISH probe labeling, PMA-qPCR, and other live-dead cell discrimination techniques all require high-quality single-cell extraction from the microbial community. However, on the one hand, when processing solid complex matrix (such as Daqu, soil) samples, they usually contain aggregated particles, extracellular polymeric substances (EPS) and complex microbial communities, making it more challenging to effectively extract microbial communities. On the other hand, according to their metabolic activity, active microbial communities can actually be divided into two categories: 1) microorganisms with true metabolic activity phenotype. 2) potential active functional presenters in a dormant state, such as spores or spores. Although this type of microbial community is in a low metabolic activity state, they do not completely lose biological activity, but can quickly restore metabolic activity and re-participate in the performance of ecological functions under appropriate environmental conditions. For example, in soil and fermented foods, many microorganisms often exist in the form of dormant bodies, which can play an important role when environmental conditions change and maintain functional stability in soil and fermentation processes. As a group with potential recovery ability, dormant bodies (spores / spores) interfere with single-cell-based live-dead cell discrimination techniques and limit researchers' comprehensive understanding of active trophozoite microbial communities in situ systems. SUMMARY

[0005] In view of the existing problems, a high-efficiency and low-loss Nycodenz-Percoll extraction strategy is developed in this paper, aiming to solve the problems of low extraction efficiency and structural bias of microbial community extraction in traditional methods. This method not only effectively distinguishes between trophozoites and dormant bodies, but also effectively identifies active microorganisms, thereby more comprehensively reflecting the diversity and ecological function of microbial communities. In particular, the Nycodenz-Percoll method can separate microbial populations in different states while maintaining microbial activity. The existence of such microbial communities means that the potential activity of microbial communities is much more complex than what is seen on the surface, which enables the microbial community in the ecosystem to maintain its flexible metabolic potential when facing extreme environments. Therefore, through this strategy, researchers can more accurately capture the dynamic changes of microbial populations in different physiological states and provide more reliable data support to promote the development of microbial ecology and environmental microbiology research.

[0006] The present application provides a high-efficiency active microbial community extraction strategy and a non-destructive dormant body cell enrichment method.

[0007] The present application provides a method for simultaneously enriching trophozoites and dormant bodies, which comprises the following steps:

[0008] (1) adding a cell extraction solution to the sample, mixing and centrifuging, collecting the supernatant; adding a cell extraction solution to the precipitate, mixing and centrifuging, taking the supernatant; combining the two supernatants;

[0009] (2) centrifuging, filtering the combined supernatant obtained in step (1), high-speed centrifuging the supernatant, discarding the supernatant, resuspending the precipitate after high-speed centrifugation, and obtaining a bacterial suspension;

[0010] (3) adding the bacterial suspension obtained in step (2) to the upper layer of an iohexol solution with a concentration of 40-80%, high-speed centrifuging, taking the lower layer of the separated solution after centrifugation, adding a buffer solution to the solution and high-speed centrifuging, resuspending the obtained precipitate, and obtaining a resuspension;

[0011] (4) configuring a double-layer concentration gradient Percoll separation solution: adding a 20-50% concentration Percoll solution to the upper layer containing 70-90% concentration Percoll, and obtaining a double-layer concentration gradient Percoll separation solution;

[0012] (5) adding the resuspension obtained in step (3) to the upper layer of the double-layer concentration gradient Percoll separation solution obtained in step (4), high-speed centrifuging, and obtaining the precipitate as the dormant body;

[0013] (6) adding a sodium chloride solution to the supernatant after centrifugation obtained in step (5), high-speed centrifuging, and taking the precipitate as the trophozoite.

[0014] In an embodiment of the present application, in step (1), the cell extract is prepared by adding Tween 20 to a system containing a final concentration of 10-50 mM sodium pyrophosphate, so that the mass fraction of Tween 20 is 0.1-1%, and the solvent in the system is a PBS buffer solution with a pH of 7.0-7.8;

[0015] In an embodiment of the present application, in step (1), the cell extract is mixed according to a mass-volume ratio of 1:1-2;

[0016] In an embodiment of the present application, in step (1), the mixing is performed by using a vortex shaker under the following conditions: 1500-3500 rpm, 20-45 min;

[0017] In an embodiment of the present application, in step (1), the centrifugation is performed at 10-50 rpm for 1-2 min.

[0018] In an embodiment of the present application, in step (2), the centrifugation of the combined supernatant is performed at 10-50 rpm for 1-2 min;

[0019] In an embodiment of the present application, in step (2), the high-speed centrifugation is performed at 8000-15000 rpm for 2-8 min;

[0020] In one embodiment of the present application, in step (2), the precipitate is resuspended with PBS sterile buffer.

[0021] In one embodiment of the present application, in step (3), the high-speed centrifugation condition after adding the bacterial suspension to the upper layer of the iohexol solution is 10000-15000 rpm for 20-50 min.

[0022] In one embodiment of the present application, in step (3), the high-speed centrifugation condition after adding an equal amount of PBS buffer to the solution is 8000-12000 rpm for 2-5 min.

[0023] In one embodiment of the present application, in step (4), the Percoll concentration of the upper layer is 40-45%, preferably 40%, and the Percoll concentration of the lower layer is 80-85%, preferably 80%.

[0024] In one embodiment of the present application, in step (5), the high-speed centrifugation condition is 8000-14000 rpm for 2-10 min.

[0025] In one embodiment of the present application, in step (6), the concentration of sodium chloride is 0.75-0.90%, and the amount of sodium chloride added is 0.8-1.5 times the volume of the supernatant.

[0026] In one embodiment of the present application, in step (6), the high-speed centrifugation condition is 8000-12000 rpm for 2-10 min.

[0027] In one embodiment of the present application, the sample includes, but is not limited to, Daqu, soil, fermented grains, solid-state fermented products, cheese, cocoa fermented products, sludge, sauce, tea, etc.

[0028] The method for efficiently identifying the composition of active and dormant bacterial populations provided by the present application includes the following steps:

[0029] 1) Take the sample in a 50 ml sterile centrifuge tube, add A1 cell extract, vortex, and low-speed centrifuge to collect supernatant 1;

[0030] 2) Continue to add A1 cell extract to the obtained bacterial precipitate, stir, and low-speed centrifuge to collect supernatant 2;

[0031] 3) Combine supernatant 1 and supernatant 2 to obtain combined supernatant 3. After low-speed centrifugation of the combined supernatant 3, filter the supernatant liquid with a 100 μm sterile filter screen, taking care not to disturb the bottom precipitate, and collect supernatant 4.

[0032] 4) Cell pellet: After centrifugation of the supernatant 4, discard the supernatant and collect the cell pellet (target microbial community) at the bottom. Resuspend the cell pellet in 5 ml of 1x PBS buffer to obtain a bacterial suspension.

[0033] 5) N purification: Prepare an iodixanol solution with a concentration of 80%, take 5 ml and place it in a 15 ml sterile centrifuge tube. Carefully add the bacterial suspension obtained in step (4) to the upper layer of the iodixanol solution using a sterile pipette. Centrifuge at 14,000 x g for 30 min. The sterile centrifuge tube will exhibit a typical stratification (fermented food products are typically 3 layers: upper, middle, and lower, soil: 1 layer)

[0034] 6) Carefully pipette the typical stratification band above the solution. Among them, the lower band of the fermented food sample is the most obvious (dark brown), which is rich in abundant microbial flora. Add an equal amount of 1x PBS buffer to the centrifuge tube containing the lower layer solution and centrifuge at 12,000 x g for 2 min. Discard the supernatant and resuspend the pellet with 500 μl of 1x PBS buffer.

[0035] 7) P purification: Prepare a double-layer concentration gradient Percoll solution, add 80% Percoll to the bottom layer and 40% Percoll to the upper layer. Slowly add the resuspended bacterial solution from step (6) to the upper layer of the Percoll solution and centrifuge at 12,000 x g for 5 min. Carefully pipette the upper solution into a sterile 15 ml centrifuge tube, taking care not to pipette the pellet. Continue to add 10 times the volume of 0.85% sodium chloride solution to the 15 ml centrifuge tube. Thoroughly disrupt the density gradient layer to facilitate the recovery of the bacterial pellet by centrifugation.

[0036] 8) Transfer the centrifuged bacterial pellet to a 2 ml sterile centrifuge tube and wash it with 0.85% sodium chloride solution for 2-3 times. Dilute the sample with 0.85% sodium chloride solution to OD = 0.3, take 500 μl and perform Syto9 / PI fluorescent staining.

[0037] 9) After the sample is treated according to the dye instructions, take 300 μl of the stained sample and add it to the absolute count microsphere tube for absolute quantification of the active bacterial community.

[0038] 10) Take an appropriate amount of the bacterial solution from the different layers of step (6) and the cell pellet after Percoll treatment in step (7) for plate culture to verify the cultivable effect.

[0039] The environmental sample mentioned in step 1) is 3g, and before treatment, the sample is ground and treated by 0.6mm screening; the amount of A1 cell extract is 30ml, the rotation speed of vortex is 2000rpm, and the vortex time is 40min. The A1 cell extract added in step 2) is 30ml. The low-speed centrifugation condition is 4℃, 20xg, 2min. The high-speed centrifugation condition is 12000xg, 5min. The preparation of 80% iohexol solution: weigh 4g of iohexol and place it in a 50ml sterile centrifuge tube, add 5ml of sterile water, and vortex thoroughly to dissolve. The preparation of double-layer Percoll density layer (80%-40%) uses 100% Percoll stock solution, which is diluted to 80% and 40% concentrations respectively. The live and dead fluorescent dye is purchased from Fushen Biology (cat. FS4005-40T). The absolute count microsphere tube BD Trucount Absolute Count Tubes is purchased from Becton Dickinson (cat. CNI20-A0013).

[0040] The application also provides the use of the above method in extracting single cells in a complex bacterial flora.

[0041] The application also provides the use of the above method in a kit for preparing single cells in a complex bacterial flora or in vegetative-hypersomolality separation or live cell detection.

[0042] Beneficial effects

[0043] The method can efficiently and non-destructively extract microbial flora from a complex matrix, and combine a live / dead staining reagent for physiological state identification. At the same time, the method further combines Percoll density gradient centrifugation for testing, which can further separate part of the hypersomolality and vegetative cells for secondary separation, thereby achieving the purpose of efficient and fractional purification. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 : Effect diagram before and after iohexol treatment.

[0045] Figure 2 : Effect diagram of extracting different types of single bacteria by Percoll solution.

[0046] Figure 3 : Effect diagram of Percoll treatment after mutual combination of single strains.

[0047] Figure 4 : Effect diagram of Percoll treatment after mutual combination of double strains.

[0048] Figure 5 : Effect diagram of Percoll treatment after mutual combination of three strains and mixed strains.

[0049] Figure 6 : The standard workflow for processing environmental samples using Nycodenz-percoll.

[0050] Figure 7 Soil samples were used to quantitatively compare the results of classical methods and Nycodenz-percoll processing.

[0051] Figure 8 The universality and advantages of Nycodenz-percoll compared to classic methods.

[0052] Figure 9 The difference in microbial composition before and after extraction was evaluated based on the composition of the microbial community.

[0053] Figure 10 Evaluation of Nycodenz-percoll content loss during extraction based on internal control fluorescent strain.

[0054] Figure 11 The effect of Percoll purification on the enrichment efficiency and cell recovery rate of dormant cells was quantitatively evaluated based on simulated bacterial communities. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] The high-temperature Daqu sample involved in the following embodiments was obtained from a liquor producer in Maotai Town, Renhuai District, Zunyi City, Guizhou Province, abbreviated as Daqu; the liquor sample involved was obtained from a liquor producer in Maotai Town, Renhuai District, Zunyi City, Guizhou Province, abbreviated as JP; the soil sample was taken from the flower bed soil of Jiangnan University in Binhu District, Wuxi City, Jiangsu Province, abbreviated as Soil.

[0057] The reagents and equipment involved in the following examples:

[0058] Reagents: Iohexol, sterile water, sterile A1 extract, 1×PBS sterile buffer, 10×PBS sterile buffer, 0.85% sodium chloride solution, sterile 100%, sterile 80%, and 40% Percoll solutions.

[0059] Equipment: 2ml / 5ml sterile droppers, 50ml / 15ml / 2ml enzyme-free centrifuge tubes, vortex mixer, handheld disperser (commonly known as: egg beater), 0.22μm sterile water filter head, sterile 100μm cell sieve.

[0060] Cultivation of strains involved in the following examples:

[0061] Obtaining of mold spores:

[0062] Firstly, the purification method for mold is described as follows: the spores cultured on PDA plate for 4-5 days are washed with 20 ml sterile water and scraped off, appropriate amount of glass beads is added, and the mixture is placed at 4°C and shaken at 200 rpm for 1 h; then, the bacterial solution is centrifuged at 8000 rpm for 5 min at 4°C, the precipitate is collected, washed once with 20 ml sterile water, and then centrifuged to harvest the bacterial body; the bacterial body is resuspended with 3 ml sterile water, filtered with Miracloth (cat. 475855-1R), the obtained filtrate is centrifuged, the supernatant is discarded, and 0.5 ml 0.1% Triton X-100 is added for storage in a 4°C refrigerator.

[0063] Obtaining of bacterial spores:

[0064] The purification method for bacteria is described as follows: the spores cultured on spore-producing F plate (consisting of 1% glucose, 0.1% L-glutamic acid, 0.05% yeast extract, 0.5% KH2PO4, 0.1% (NH4)3PO4, 0.02% MgSO4, 0.01% NaCl, 0.005% CaCl2, 0.0007% MnSO4, 0.001% ZnSO4, and 0.001% FeSO4) for 7 days are washed with 20 ml sterile water and scraped off, appropriate amount of glass beads is added, 20 mg / ml lysozyme 200 μl is added to 20 ml spore suspension, and the mixture is shaken at 37°C and 200 rpm for 1 h; then, the mixture is placed in a 80°C water bath for 0.5 h to inactivate the vegetative cells. The bacterial solution is centrifuged at 8000 rpm for 5 min at 4°C, the precipitate is collected, resuspended with 5 ml 0.05% SDS, and ultrasonically centrifuged for 10 min; then, the mixture is centrifuged at 8000 rpm for 5 min, the precipitate is washed with 5 ml water for 3 times, the precipitate is then resuspended to 3 ml, filtered with filter cloth, the filtrate is collected, centrifuged to obtain the precipitate, and 0.5 ml 0.1% Triton X-100 is added for storage in a 4°C refrigerator.

[0065] Cultivation of bacterial cells:

[0066] Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus megaterium strains are respectively inoculated into LB solid medium, and cultured at 37°C for 12 h to prepare seed liquid; the prepared seed liquid is respectively inoculated into LB liquid medium by picking single colonies of the seed culture medium, and cultured at 37°C for 12 h to prepare bacterial solution.

[0067] Fungal spore culture:

[0068] Lichtheimia ramosa, Rhizopus microsporus and Aspergillus oryzae 3.042 were streaked onto PDA solid medium and incubated at 30°C for 96 h to prepare seed liquid; the prepared seed liquid was scraped with sterile water to obtain spore suspension; Miracloth (cat. 475855-1R) was used to separate mycelium and spores for subsequent experiments.

[0069] Yeast spore culture:

[0070] Rhodotorula mucilaginosa, Saccharomyces cerevisiae and Candida parapsilosis were streaked onto YPD solid medium and incubated at 30°C for 48 h to prepare seed liquid; the prepared seed liquid was inoculated into YPD liquid medium and incubated at 37°C for 12 h to prepare bacterial liquid.

[0071] P purification verification experiment:

[0072] At present, the classical dormant body enrichment method is widely used in the structural analysis of community ecology, especially in the isolation of dormant bodies such as spores or spores in microbial communities. This method has strong universality. In view of the fact that dormant bodies in complex microbial communities (such as spores or spores) have stronger cell walls and stronger tolerance, they can resist high temperature, strong alkali, SDS and other treatments. The vegetative cells, especially non-spore bacteria or fungi (such as yeast and filamentous fungi), have relatively fragile cell walls and membrane structures, which are specifically destroyed under strong physical and chemical factors, and finally lose metabolic activity. Although the classical method can effectively destroy the vegetative cells in the community by adjusting the treatment time, reaction temperature and treatment liquid concentration. However, these traditional methods still have some problems, especially when the solution concentration and incubation time are not properly set, which will cause the survival rate of dormant bodies to decrease significantly, and cause irreversible damage to the subsequent culturable process.

[0073] To address the aforementioned issues, density gradient centrifugation, as an alternative strategy, provides a more gentle and efficient separation method. By exploiting the distribution differences of cells in different density media, density gradient centrifugation can effectively separate dormant bodies from other cells without exerting excessive physical or chemical pressure on microbial cells. Compared to traditional chemical treatment methods, density gradient centrifugation can more accurately isolate target dormant bodies, avoiding cell damage that may be caused by chemical treatment and greatly reducing species loss due to improper handling. Therefore, density gradient centrifugation has obvious advantages in dormant body enrichment, especially in research that requires the preservation of cell activity and maintenance of ecological diversity, exhibiting higher separation efficiency and lower handling risk. Among them, Nycodenz density gradient centrifugation, as a classic extraction strategy for soil sample pretreatment, can effectively separate microflora from complex matrices. Therefore, in the early stage of the experiment, this paper takes Nycodenz density gradient centrifugation as an example to verify whether it has the ability to target enrichment of dormant bodies in complex bacterial flora. However, the results of the simulated bacterial flora show that Nycodenz does not have the performance of targeting enrichment of dormant bodies (spores and spores) in complex systems. In response to this problem, this paper further attempts to introduce Percoll density gradient centrifugation. As a more accurate and efficient separation strategy, Percoll solution has shown excellent performance in effectively enriching dormant bodies in complex bacterial flora, which has successfully overcome the limitations of traditional Nycodenz methods in the separation process. In particular, in the process of actual sample processing, the combination of the two can successfully separate vegetative cells and dormant bacterial flora, thus providing an ideal solution for fine research on the structural composition of the community.

[0074] The detection method involved in the following examples is as follows:

[0075] A. Detection of cell recovery rate

[0076] In order to ensure that the bacterial solution is uniformly adsorbed to the surface of the filter membrane, about 5 ml of sterile PBS solution is placed in the filtration container, and then an appropriate concentration of bacterial solution is added and stirred thoroughly before filtration, so that the bacterial solution is uniformly adsorbed to the surface of the black polyester carbonate membrane (MemberSpace) with a pore size of 0.22 μm and a diameter of 13 mm. Then a small amount of sterile water is used to rinse the membrane to prevent PBS from crystallizing and interfering with microscopic observation. The black polyester carbonate membrane obtained is clamped onto a glass slide with tweezers, 10 μl of anti-fluorescence quencher is added to cover the surface of the filter membrane, and a cover glass is carefully pressed on to avoid air bubbles.

[0077] The fluorescence microscope was used to adjust the channel for fluorescence image acquisition. ImageJ software was used for noise removal, background optimization and other series of processing, and then counting. According to the description of Yang et. al, 16 images were randomly collected at different positions of each filter (i). After taking pictures, the total number of microorganisms in the field of view of each filter was counted a, which needed to meet:

[0078]

[0079] The total number of microorganisms on each filter was estimated:

[0080]

[0081] Where m is the area of the field of view, a is the total number of microorganisms in the 16 fields of view on the filter, and d is the inner diameter of the funnel contacting the filter. The number of microorganisms in the original sample x = c / iV was estimated, where V is the volume of the original sample calculated to be filtered onto a single filter, and i is the number of parallel groups.

[0082] Recovery rate = (N-P method after treatment cell concentration / initial added strain concentration) x 100%.

[0083] B. Detection of cell dormancy body residual rate

[0084] Spores were counted according to the method mentioned in the "detection of cell recovery rate" described above.

[0085] It should be noted that the collected spore-containing filter membrane is immersed and dyed using the classic spore dyeing method, and then washed with sterile water several times to remove the excess dye fluorescence interference. Finally, the fluorescence count is carried out under the mCherry channel, and the spores are bright red under the fluorescence microscope. Under the observation of 100X oil lens, spores can be effectively distinguished from artificial flora by their size and color. Secondly, mold spores can be effectively distinguished from artificial flora and spores under 60x objective lens according to their color and shape differences.

[0086] Therefore, this paper selects the blood cell counting plate to count the number of spores.

[0087] Example 1: Explore the separation efficiency of dormancy body under different purification strategies

[0088] Simulation of flora introduction:

[0089] To test the effectiveness of the method, as shown in Table 1, 3 strains of yeast, 3 strains of bacteria, and 3 strains of mold were selected to form a mixed microbial population, and their vegetative cells and corresponding spores were obtained. A simplified strategy was used to simulate the composition of the microbial population structure in the environment sample. First, the present application "qualitatively" evaluated the difference in the effect of iodixanol and Percoll density gradient centrifugation on the separation of spores in complex microbial populations.

[0090] Table 1: Representative species names and corresponding experimental materials (vegetative cells / spores) involved in the experiment

[0091]

[0092] First, the present application tests the enrichment effect of iodixanol on spores in complex microbial populations.

[0093] Specifically as follows:

[0094] 1. Effect of iodixanol method enrichment:

[0095] The specific method is:

[0096] (1) Prepare an iodixanol solution with a concentration of 80%, and take 1 ml and add it to a 1.5 ml centrifuge tube;

[0097] (2) Use a spectrophotometer to measure the concentration of the cultured bacteria (OD 600nm ), and adjust the OD 600nm of the bacteria and yeast cells to about 0.5;

[0098] (3) Add 50 μl of the bacterial solution from step (2) to the top of the 1 ml iodixanol solution from step (1), and centrifuge at 14000 x g for 30 min;

[0099] 2. Single bacteria level - Percoll method enrichment effect

[0100] The present application also verifies the effect of Percoll solution treatment at the single bacteria level (Panel 16-29), which is divided into three groups: bacteria group, yeast group, and mold group.

[0101] The specific method is:

[0102] (1) Take 80% (500 μl) and 40% (500 μl) concentration Percoll solution, and place 500 μl of 80% concentration Percoll solution at the bottom. Then, slowly add 500 μl of 40% Percoll solution;

[0103] (2) Slowly add 50 μl of bacterial solution (OD600nm After the centrifugation, the supernatant was collected and the cells were washed with 1 ml of PBS. The washing step was repeated twice. The cells were then resuspended in 1 ml of PBS and the concentration was adjusted to 1 x 108cells / ml. The cells were then centrifuged at 14000 x g for 5 min.

[0104] The results are shown in Figure 2

[0105] Panels 16-18 tested the position of three types of yeast vegetative cells after Percoll density centrifugation.

[0106] Panels 19-23 tested the separation effect of 3 types of vegetative cells and 2 types of bacterial spores.

[0107] Panels 24-29 tested the separation effect of 3 types of spores and 3 types of vegetative cells.

[0108] In summary, the results are shown by induction Figure 1 and Figure 2

[0109] Figure 1 : Effect diagram before and after iohexol treatment, A-All (before iohexol treatment), and A1-All1 (after iohexol treatment). The specific groups contained are as follows: A- Bacillus amyloliquefaciens (spore); B- Aspergillus oryzae 3.042 (spore); C- Bacillus amyloliquefaciens and Bacillus velezensis (spore); D- Aspergillus oryzae 3.042 and Rhizopus microsporus (spore); E- Bacillus amyloliquefaciens and Aspergillus oryzae 3.042 (vegetative cell); F- Bacillus velezensis and Rhizopus microspores (spore and spore); All- vegetative cell and spore system containing all the above species.

[0110] Figure 2 ​​: The effect diagram after single-strain Percoll treatment (Panel 16-29). The total is divided into three groups: yeast group, bacterial group, and mold group; the designed species in each group are as follows: (i) Yeast group: Rhodotorula mucilaginosa- mycelium (16), Saccharomyces cerevisiae- mycelium (17), Candida parapsilosis- mycelium (18); (ii) Bacterial group: Bacillus amyloliquefaciens- mycelium (19) / spore (22), Bacillus velezensis- mycelium (20) / spore (23), Bacillus megaterium- mycelium (21); (iii) Mold group: Lichtheimia ramos- mycelium (24) / spore (27), Rhizopus microsporuss- mycelium (25) / spore (28), Aspergillus oryzae 3.042- mycelium (26) / spore (29).

[0111] From the qualitative evaluation: the iohexol method only has a certain spore removal effect on Bacillus amyloliquefaciens, and basically has no effect on the rest. Therefore, this method cannot achieve effective removal of spores and spores. In contrast, Percoll can effectively achieve targeted separation of spores and spores, and yeast cells are generally concentrated in the middle layer, and mold mycelium is mainly in the upper layer. The true realization of the precise treatment of different groups of cells.

[0112] 3. Combination verification experiment of Percoll density gradient centrifugation method

[0113] Based on the above steps, the present application introduces the Percoll density gradient centrifugation method into the subsequent verification process, and explores the correlation between the complexity of the microbial community (single bacteria-dual bacteria-mixed bacteria) and the treatment ability of Percoll, as follows:

[0114] A. Single-strain combination experiment:

[0115] (1) Take 80% (500 μl) and 40% (500 μl) concentration of Percoll solution, place 500 μl of 80% concentration of Percoll solution at the bottom, then slowly add 500 μl of 40% concentration of Percoll solution;

[0116] (2) Select three representative strains for single-strain level combination test, slowly add 50 μl of bacterial solution (OD 600nmat 14000 x g for 5 min;

[0117] The results are shown in Figure 3

[0118] Figure 3 : The effect diagram of single strain combination after Percoll treatment (Panel 1-5). According to the addition principle of Panel 1-5 in the table, different types of bacteria or dormant bodies were added to S. cerevisia to conduct mixed tests.

[0119] The results show that the density of yeast cells is moderate, mostly in the middle layer. In contrast, the density of mycelium is relatively small and can be captured in the upper layer of Percoll solution. Spores and spores have a larger density and are effectively separated from the vegetative cells.

[0120] B, double bacteria combination experiment

[0121] Similarly, the operation is the same as the single bacteria experiment. The difference is that this study gradually increases the complexity of the system. From the original selection of a single strain in each category for combination test, to the selection of 2 strains in each category for combination test, to verify whether the increase of population complexity will lead to the decline of Percoll processing efficiency.

[0122] (1) Take 80% (500 μl) and 40% (500 μl) concentration of Percoll solution, place 500 μl of 80% concentration of Percoll solution in the bottom layer, then slowly add 500 μl of 40% concentration of Percoll solution;

[0123] (2) Slowly add 50 μl of bacteria solution (OD 600nm at 14000 x g for 5 min;

[0124] The specific groups are as follows:

[0125] Figure 4 ​: the effect diagram of the combination of two strains after Percoll treatment (Panel 6-10). Based on Saccharomyces cerevisia and Rhodotorula mucilaginosa, different types of bacteria or dormant bodies are added to S. cerevisia and R. mucilaginosa respectively according to the addition principle of Panel 6-10 in the table for mixed test.

[0126] As shown in Figure 4 Panel 6-10, the specific details are as follows: taking the yeast group as the initial reference (Panel 10), the yeast species in the system is upgraded from 1 to 2 mixed. At the same time, other types of bacteria are gradually introduced into the system to increase the complexity of the system. Panel 6 represents the introduction of bacterial vegetative cells into the original (Panel 10) simple system; Panel 7 introduces bacterial spores into the original system; Panel 8 introduces mold vegetative cells into the original system; and Panel 9 introduces mold spores into the original system.

[0127] From the cell stratification results, it can be seen that the Percoll method has good separation effect on spores and spores, for example: Panel 7 and Panel 9 show good spore and spore removal effect;

[0128] C, three-strain combination experiment:

[0129] In order to simulate more complex microbial community composition, the present application continues to try to upgrade the species in a single category from 1 to 3. Panel 11-15 groups represent a higher complexity of mixed microbial community.

[0130] (1) 80% (500 μl) and 40% (500 μl) concentration of Percoll solution is taken, 500 μl of 80% concentration of Percoll solution is placed in the bottom layer, then 500 μl of 40% Percoll solution is slowly added;

[0131] (2) 50 μl of bacteria solution (OD 600nm 0.5 or so) is slowly added to the upper part of the 1 ml Percoll solution obtained in step (1) respectively, and centrifugation is carried out at 14000xg, 5 min;

[0132] The specific groups are as follows:

[0133] Figure 5: the effect diagram of three strains and mixed strains after Percoll treatment (Panel 11-All). Based on the three types of yeast mixed liquid as a reference, according to the addition principle of Panel 11-All in the table, different types of cells or dormant bodies of different types were added to the mixed liquid of three types of yeast respectively, and mixed test was carried out.

[0134] As shown in Figure 5 , the All-group in the three-strain combination verification result includes the vegetative cells of all strains in Table 1 and their corresponding dormant bodies, which together form a complex group.

[0135] From the separation results, the liquid in the Panel 11-15 group was separated by Percoll cell liquid, and the groups with spores or spores in Panel 12, 14 and All group showed good analysis effect, which further verified the excellent performance of Percoll in separating dormant bodies and vegetative cells.

[0136] 4. Optimization of Percoll purification experiment conditions:

[0137] In order to verify the influence of Percoll concentration gradient on the separation effect, the concentration gradient test was tried in this paper.

[0138] (1) The specific operation is the same as in Example 1, except that in the single density gradient Percoll experiment, the volume fraction of 90% Percoll solution is used instead of 80% Percoll, and the concentration difference between 90% and 80% Percoll is tested. The results show that there is no obvious difference between the two in separating dormant body bacteria. Therefore, the 80% Percoll concentration gradient is selected as the supporting medium of the bottom layer.

[0139] (2) This paper introduces the test of 80%-40% and 80%-60% double density into the system, and the specific steps are the same as in the example, except that the difference of the upper layer Percoll density (concentration) is set to 40% and 60% respectively. The results show that the 80%-60% density layer will cause the phenomenon of unclear layering in the separation process. Therefore, the final double density gradient layer is set to 80%-40% for subsequent optimization test.

[0140] (3) In the centrifugation time of Percoll, this paper respectively uses 7000xg, 10min centrifugation speed and 14000xg, 5min parameters for comparison after separation. The specific steps are the same as in Example 1 under "2, single strain level - Percoll method for enrichment effect", except that the centrifugation speed and time are changed; the results show that there is no significant difference between low speed and long time and high speed and short time.

[0141] Therefore, the above results show that in the pure dormant body bacterial solution, compared with the bacterial sediment obtained by low speed centrifugation, 14000xg, 5min can achieve better separation effect.

[0142] Example 2: Targeted enrichment of dormant bacterial flora using improved iodixanol purification strategy

[0143] Reference Figure 6 Operation flow test:

[0144] 1. Prepare cell extraction solution: prepare a mixture containing 25mM sodium pyrophosphate and 0.5% Tween 20 by mass fraction in 1xPBS buffer solution (pH: 7.2) as solvent, adjust pH to 7.2, and pass through 0.22μm filter membrane for standby.

[0145] Weigh 3g of high-temperature Daqu sample (the sample is ground and sieved through 0.6mm) into a 50ml sterile centrifuge tube, add 30ml of the above cell extraction solution, and place it on a vortex shaker at 2000rpm for 40min; after the end, centrifuge the above sample at 20xg for 1min; obtain supernatant 1 and precipitate 1, and transfer the obtained supernatant 1 to a new 50ml sterile centrifuge tube.

[0146] 2. Add 20ml of the cell extraction solution prepared in step 1 to the precipitate 1 obtained in step 1, adjust to medium speed with a handheld disperser, stir for 1min, then centrifuge the sample at 20xg for 1min to obtain supernatant 2.

[0147] 3. Centrifuge the combined supernatant 1 and supernatant 2 at 20xg for 1min to obtain supernatant 3, and filter the obtained supernatant 3 with a 100μm cell screen to remove residual rice hulls and other low-density floating impurities to obtain supernatant 4.

[0148] 4. Place the supernatant 4 obtained in step 3 in a 50ml sterile centrifuge tube, and centrifuge the centrifuge tube at 11040xg for 5min, discard the supernatant, and obtain the sample containing cell precipitate (target bacterial flora), add 5ml of 1xPBS sterile buffer to resuspend the bacterial cells, and obtain the bacterial suspension.

[0149] 5. N purification: weigh 4 g of iohexol solution, add 5 ml of sterile water, vortex at 2000 rpm for 10 min, then mix thoroughly to obtain a uniform solution, filter sterilize with a 0.22 μm sterile filter, and obtain a sterile iohexol solution with a concentration of 80% (g / 100 ml). Take 5 ml of the above iohexol solution and place it in a 15 ml sterile centrifuge tube. Carefully add the bacterial suspension obtained in step 4 to the upper layer of the iohexol solution using a 5 ml sterile pipette. Centrifuge at 14000 x g for 30 min. The high-temperature Daqu sample will exhibit a typical upper, middle, and lower three layers in the sterile centrifuge tube.

[0150] 6. Carefully pipette the typical cell band in the centrifuge tube. The upper cell band has the highest purity and smallest density. The middle cell band has higher cell purity and moderate density. The lower band of the high-temperature Daqu sample has high bacterial content and dark color, rich in microbial flora.

[0151] Therefore, the lower layer of the most abundant species was separated and subjected to subsequent Percoll purification. After separating the lower layer solution, add an equal amount of 1 x PBS buffer and centrifuge at 12000 x g for 2 min. Discard the supernatant and resuspend the precipitate with 500 μl of 1 x PBS buffer.

[0152] 7. P purification:

[0153] (1) Prepare a sterile Percoll solution. First, prepare a 100% Percoll mother liquor (1 ml of 10 x PBS buffer added to 9 ml of Percoll stock solution). After filtering sterilization with a 0.22 μm sterile filter, take 800 μl and 400 μl of 100% Percoll solution, respectively, and add 200 μl and 600 μl of 1 x PBS buffer, respectively, to prepare Percoll solutions with concentrations of 80% and 40%, respectively.

[0154] (2) Prepare a double-layer concentration gradient Percoll separation solution.

[0155] Take a 2 ml sterile tube and add 500 μl of Percoll (80%) solution to the bottom layer. Then, carefully add 500 μl of Percoll (40%) solution slowly on top of the 80% concentration layer using a pipette to obtain a double-layer concentration gradient Percoll separation solution.

[0156] (3) Slowly add the resuspended bacteria solution from step 6 to the top of the 40% Percoll solution of the double-layered concentration gradient obtained in step (2) and centrifuge at 14000 x g for 5 min. Carefully pipette all the supernatant into a sterile 15 ml centrifuge tube, leaving the pellet at the bottom of the tube (be careful not to pipette the pellet). Add 10 times the volume of 0.85% NaCl solution to the 15 ml centrifuge tube and centrifuge at 14000 x g for 5 min. The purpose of this step is to completely destroy the density gradient layer, making it easier to recover the bacterial cells by centrifugation. The pellet obtained is the bacterial cells.

[0157] 8. Transfer the centrifuged bacterial cells obtained in step 7 to a 2 ml sterile centrifuge tube and wash 2-3 times with 0.85% NaCl solution. Dilute the washed sample with 0.85% NaCl solution to OD 600nm = 0.3 and take 500 μl of the sample for live / dead fluorescent staining.

[0158] 9. After processing according to the instructions for the complex biological dye, take 300 μl of the stained sample and add it to the flow tube for machine analysis.

[0159] 10. Take an appropriate amount of the bacterial solution from the different layers obtained in step 6 and the cell pellet after Percoll treatment in step 7 for plate culture and verification of culturability.

[0160] Comparative Example 1: Use the classic density gradient centrifugation method to test the advantages of the NP strategy compared to the classic method

[0161] The specific method is described in: Direct cell extraction from fresh and stored soil samples: Impact on microbial viability and community compositions, 2021, Soil Biology and Biochemistry.

[0162] Reagents and equipment involved: Reagents: Iohexol, 0.1% sodium deoxycholate (SD), 80% sterile iohexol solution, sterile water, 1x PBS buffer. Equipment: 2 ml / 5 ml sterile dropper, 50 ml / 15 ml / 2 ml non-enzyme centrifuge tube, vortex shaker, Waring blender (Conair TM 7012S, cat. 14-509-7G), 0.22 μm sterile water filter, sterile 30 μm cell screen.

[0163] The specific steps are as follows:

[0164] 1) Weigh 10 g of soil into 20 ml of SD solution, using a Waring blender, blend for 3 min at maximum speed, with 1 min intervals, continue incubating the sample on ice for 1 min to cool the mixture.

[0165] 2) First iodixanol treatment: Weigh 24 g of iodixanol into 30 ml of sterile water, after complete dissolution, sterilize using a 0.22 pm sterile water filter head, to obtain a sterile iodixanol solution at 80% concentration, take 20 ml of iodixanol solution at 80% concentration and add it into a 50 ml sterile centrifuge tube, slowly add 20 ml of soil slurry from step 1) on top of the iodixanol solution. Place at 4°C, 14000 x g, centrifuge for 40 min.

[0166] 3) After centrifugation, carefully collect the cell layer on top of the Nycodenz using a 5 ml pipette and transfer it to a new sterile 50 ml tube. Filter the mixed solution through a sterile filter (SmartStrainers, 30 pm pore size, cat. 130-098-458) into a new sterile Oak Ridge centrifuge tube to remove large soil debris, this filtration step does not lose cells. Centrifuge at 4°C, 14000 x g, for 15 min to pellet the soil-extracted cells. Discard the supernatant and resuspend the cell pellet in 5 ml of PBS buffer and designate as “1st round of cells”.

[0167] 4) Second iodixanol treatment: Repeat step 3) with the soil pellet obtained from the first Nycodenz purification. The cells harvested in this round are designated as “2nd round of cells”. Mix the cell pellet from the first extraction with the cell pellet obtained after the second extraction. Then, centrifuge at 12000 x g for 5 min, keeping the pellet. Wash the cell pellet three times with 1 x PBS buffer, centrifugation conditions: 12000 x g for 5 min.

[0168] 5) Transfer the centrifuged cell pellet to a 2 ml sterile centrifuge tube and wash 2-3 times with 0.85% sodium chloride solution. Dilute the sample to an OD 600nm = 0.3 with 0.85% sodium chloride solution.

[0169] 6) To quantitatively assess the recovery yield of viable bacterial population from the sample, after following the instructions of the LIVE / DEAD® Bacterial Dye kit, in this document we took 500 pl of the bacterial solution for Syto9 / PI fluorescent staining. Take 300 pl of the stained sample and add it to a BD Absolute Count Microsphere Tube for the absolute quantification of the viable bacterial population.

[0170] The results are shown in Figure 7

[0171] Figure 7 ​​: The results of the classic method and the Nycodenz-percoll treatment were quantitatively compared using soil samples. a. The differences in the results of the classic method and the present application in extracting soil environment samples are presented. First, during the first round of iodinated contrast medium extraction process, the cell band extracted by the classic method has many impurities, the band is diffuse, and the amount is small. The extraction method of the present application has no obvious impurities, the band is compact, and the amount is large. b. For the same soil sample, the results of the live and dead quantitative statistics combined with flow show that the recovery yield of the soil_B extracted by the present application is much higher than that of the classic method. c. The live and dead area division staining diagram of the soil sample treated by Nycodenz in the present application.

[0172] The results show that, compared with the classic scheme and the Nycodenz-percoll method of the present application, the cell content in the classic scheme is less, and there are obvious impurities in the band from the first round of iodinated contrast medium purification. In contrast, the N-P method of the present application can obtain higher cell purity and content. At the same time, combined with the statistical results of the absolute quantitative counting tube, the total amount of active microorganisms (4.4x 10 5 / g) obtained by the present application in the first extraction is significantly higher than that of the classic iodinated contrast medium (1.45x 10 5 / g) extraction scheme. From the flow chart, it can also be seen that this method can effectively divide the live and dead communities, and is a high-efficiency and high-purity cell extraction method.

[0173] Example 3: Test of the extraction effect of the Nycodenz-percoll separation method of the present application in different environment samples

[0174] The specific first 5 steps are the same as in Example 2, the difference is that the sample is adjusted as follows:

[0175] Sample source: Baijiu producer in Maotai Town, Renhuai District, Zunyi City, Guizhou Province, abbreviated as: Daqu(A);

[0176] The wine sample provided by the Baijiu producer in Maotai Town, Renhuai District, Zunyi City, Guizhou Province (abbreviated as: JP);

[0177] The soil sample was taken from the flower bed of Jiangnan University in Binhu District, Wuxi City, Jiangsu Province (abbreviated as: Soil);

[0178] According to the method of Example 2, the dormant body-enriched-JP-1 and the dormant body-enriched-Soil-1 were prepared;

[0179] The test results are shown in Figure 8 .

[0180] First, as shown in Figure 8 a, the effect diagram of the classic method for extracting Daqu samples;

[0181] Secondly, the test effect diagram of different environmental samples is as shown in Figure 8 b-c:

[0182] A. According to steps 1-5 in Reference Example 2, after the soil sample is treated by iohexol, only a single cell band is presented, the cell purity is high, and there is no obvious impurity interference.

[0183] B. According to steps 1-5 in Reference Example 2, after the high-temperature Daqu sample is treated by iohexol, a unique three-layer cell band is presented, and the cell purity of the sample is high, and there is no obvious impurity interference.

[0184] C. According to steps 1-5 in Reference Example 2, after the JP sample is treated by iohexol, a unique two-layer cell band is presented, the upper band of the sample has high purity, and the lower band presents dark brown.

[0185] Among them, since the upper and middle bands of the high-temperature Daqu have high purity, only the lower band is turbid and presents dark brown. The microscopic observation results show that the cell types and content of the lower layer are higher.

[0186] Finally, the sample with typical layered Daqu is analyzed in depth in this paper, and the samples extracted from each cell layer are displayed. At the same time, this paper also further purifies the brown layer (3), as shown in Figure 8 c, the effect diagram before and after P purification of the present application is presented.

[0187] Therefore, as shown in Figure 8 ;

[0188] Figure 8The universality and advantages of Nycodenz-Percoll compared to classical methods. a. Effect image of high-temperature Daqu samples after treatment with classical methods. b. Effect image of soil, high-temperature Daqu, and liquor samples after purification by the present invention Nycodenz. c. Taking a complex environment sample - high-temperature Daqu - as an example, 1-3 show the upper, middle, and lower three-layer cell solutions extracted after iohexol treatment. At the same time, Percoll treatment was performed on 3 (lower layer), visually showing the effect before and after Percoll treatment. d. Taking high-temperature Daqu as an example, the 40X micrograph corresponding to the "bacterial suspension" (Enrich) part in "step 4" of Example 2, where a large number of impurities such as starch are present and the cell ratio is low. e. 40X micrograph of soil microorganisms after N extraction in Example 2. f. 40X micrograph of soil microorganisms after P extraction in Example 2. g. Take 200 μl of the Nycodenz-percoll supernatant and precipitate (dormant bacterial colonies) prepared according to the method of Example 2 and spread them onto solid LB plates containing antifungal growth inhibitors (containing 100 μg / ml nystatin) and solid PDA plates containing antibacterial growth inhibitors (containing 100 μg / ml kanamycin), and incubate at 30°C for 1-3 days.

[0189] This invention further purified the suspension obtained from the third layer of the high-temperature Daqu (a type of Chinese liquor) using Percoll, following the same procedures as steps 6-7 in Example 2. To present the bacterial community status after different treatment stages, this invention incorporates microscopic... Figure 8 The diagrams in Figure df are shown, where Figure d represents the sample state before iohexol treatment, Figure e represents the sample state after iohexol treatment, and Figure f represents the sample state after Percoll treatment. It is clear from the diagrams that the sample treated with iohexol still has a small amount of impurities (marked by red circles).

[0190] Therefore, the Percoll purification strategy introduced in this invention can not only further improve the cell purity of the sample, but also effectively enrich dormant bacterial flora. Simultaneously, this invention describes the processing of the supernatant and precipitate of the Percoll-purified sample. 200 μl of bacterial culture was taken and inoculated onto ampicillin PDA and LB solids (n=2) using a disposable plating stick. The plating results showed that the enriched bacterial cells obtained by this method have metabolic activity and can be revived under certain conditions for subsequent culturing.

[0191] Comparative Example 2: The effect of the classic iodhexol separation method on the extraction of high-temperature Daqu (a type of Chinese liquor).

[0192] The specific operation is the same as in Comparative Example 1;

[0193] Sample source: Baijiu producers in Maotai Town, Renhuai District, Zunyi City, Guizhou Province, abbreviated as: Daqu;

[0194] Test results: as shown in a of Figure 8 It can be clearly seen that, without reference to the foregoing treatment process of the present application, the use of the classic iodixanol extraction method in the first extraction process will result in the inability to clearly identify the location of the cell layer (1), even if the second round of iodixanol extraction is experienced, the classic method still has the problem of unclear layering and excessive impurities in the extraction of high-temperature Daqu. This to some extent limits the application of Nycodenz in high-temperature Daqu. Therefore, the present application needs to try to explore new pretreatment strategies to cope with complex high-temperature Daqu fermentation samples.

[0195] Example 4: Evaluation of the structural changes of the microbial community at different processing stages based on high-throughput sequencing

[0196] The specific operation is the same as in Example 2, except that the sample at each key stage is subjected to amplicon sequencing after processing;

[0197] Sample source: high-temperature Daqu sample (abbreviation: Daqu) provided by a liquor manufacturer in Maotai Town, Renhuai District, Zunyi City, Guizhou Province;

[0198] The specific steps are as follows:

[0199] First, weigh 3g of the sample, and use conventional DNA extraction means for amplicon sequencing (named Initial-A).

[0200] Second, prepare the sample according to steps 1-4 of Example 2 (bacterial suspension, named Enrich-B), then obtain Nycodenz (Nycodenz-C) according to step 5 of Example 2, and continue to obtain the filtered sample of Percoll (precipitate obtained in step 7, named NP-D) in step 7 of Example 2 for amplicon sequencing.

[0201] The sample processing process at stage C-D is as follows: place the enriched liquid sample on a sterile filter membrane containing 0.22μm, use a vacuum negative pressure filtration device to perform filtration, and send the enriched sample to Beijing Nuohexuan Sequencing Company for subsequent quality control and machine sequencing. Use universal sequencing primers 338F-806R (bacteria) and ITS1F-ITS2R (fungi) for testing.

[0202] The results are shown in Figure 9 ;

[0203] Figure 9: Based on the composition of the bacterial flora structure to evaluate the difference between before and after extraction. a-c figure presents the structural changes of bacterial community, d-f figure presents the composition changes of fungi. From c, f two figures can be obviously seen that the treatment before and after will not cause the obvious deviation of the flora structure. Take a-c figure as an example for further explanation: a. represents the genus level species dilution curve after different treatment methods, with the increase of sequencing reads, the corresponding sobs index is higher, which means the extracted species is more. b. presents the flora structure difference after four kinds of treatment methods in the form of scatter plot, the closer the distance between the groups, the more similar the microbial composition. c. presents the species composition of the flora from the perspective of the top 20 dominant species.

[0204] The results show that:

[0205] 1) First, combined with the bacterial species dilution curve a figure, it can be seen that the species diversity of bacteria is the highest in the Enrich stage.

[0206] 2) From the overall difference figure of community structure b, it can be seen that the initial flora structure is obviously different from the flora structure of the samples after the enrichment process (B-D), but the flora samples B / C / D after enrichment have similar flora structure characteristics;

[0207] 3) At the same time, in the composition of dominant species, there is no significant fluctuation in the composition and content of the sample before and after extraction. Similarly, in the composition of fungi, it can be seen that the samples treated by Nycodenz and Nycodenz-percoll have higher species diversity, which shows that the enrichment degree of fungi is obviously improved after density gradient centrifugation.

[0208] 4) At the same time, in the flora structure and composition, it can be seen that there is no significant fluctuation before and after the treatment, which further shows that this method can achieve high recovery at the whole flora level and avoid the structure loss caused by the purification process.

[0209] Example 5: Quantitative evaluation of Nycodenz-percoll strategy based on simulated flora cell recovery rate and cyst residual rate

[0210] In order to verify the treatment effect of Nycodenz-percoll, the present application will use Daqu, wine dregs and soil samples to prepare cysts according to the method of example 2;

[0211] From the angles of recovery rate and cyst residual rate, the treatment performance of Nycodenz-percoll is evaluated. From the angle of recovery rate, combined with the flow Figure 6 chart, the present application introduces internal reference strains with color identification markers to the step of "precipitate 2 resuspension (bacterial suspension obtained in step 4 of example 2)" for the evaluation of recovery rate.

[0212] Firstly, three different environmental samples were selected: Daqu, Jiupei and soil for testing. The recovery efficiency of the N-P method was quantitatively evaluated based on the addition of internal standard strains.

[0213] Different color fluorescent strains constructed by the research group were used as internal reference strains for testing: Escherichia coli pHY300-P shuttle09-egfp (green) and Saccharomyces cerevisiae TN124-nab2-mcherry (red).

[0214] The specific operation is as follows: weigh 3g of environmental sample, immerse according to the cell extraction process, count the fluorescent internal reference strains cultured to the logarithmic growth phase under a microscope, adjust the initial addition concentration of fluorescent E. coli to 6.33x10 9 cfu / ml, and the fluorescent yeast to 1.56x10 9 cfu / ml, respectively, take 50ul and 100ul, add to the resuspended cell suspension after enrichment, and place on a vortex shaker for 20min for subsequent cell counting.

[0215] The results are shown in Figure 10

[0216] Figure 10 Evaluation of Nycodenz-percoll content loss in the extraction process based on internal reference fluorescent strains. In Example 5, the recovery rate was evaluated at the corresponding stage after treatment with iohexol (Nycodenz) and Percoll solution. DAPI dye was used to count the total number of cells in various environmental samples. During the process, the active internal reference fluorescent strains were used as the evaluation standard for recovery rate, and the recovery rate determination between samples was repeated three times.

[0217] The results show that the cell recovery rate evaluation results involved in the Nycodenz process in the present application show that this method is more friendly to bacterial recovery, with a recovery efficiency of more than 81.39%. The recovery effect of soil fungi is relatively poor, but still maintains a cell recovery rate of more than 58.98%. Secondly, in the further Percoll purification process, the recovery rate of bacteria still maintains a good level of more than 72.02%.

[0218] It should be noted that the Jiupei contains a large number of yeast cells, which may cause a large number of bacterial cells and dormant cells to be precipitated after Percoll treatment, resulting in a large cell loss rate of Jiupei samples at the fungal level. This result is closely related to the characteristics of the microbial community composition of the sample.

[0219] ​Secondly, from the removal effect evaluation of the dormant body, this paper introduces the spore residual rate for reference.

[0220] Two strains of spores and two types of spores with obvious differences in morphology and color were selected as internal reference dormant bodies for testing. During the period, spores, fluorescent internal reference strains were counted by fluorescence microscopy, mold spores and yeast were counted by blood cell counting plate. Mold mycelium was tested by scraping the mycelium after plate coating. Finally, the total amount of spores and spores added was adjusted to 1.0-2.5x 10 7 cfu / ml, and the amount of vegetative cells added was 2.0-2.5x 10 8 cfu / ml.

[0221] As Figure 11 shown:

[0222] Figure 11 : Based on the simulation of the quantitative evaluation of the influence of Percoll purification on the enrichment efficiency of dormant bodies and the recovery rate of bacterial cells. In Example 4, the present application combined the three combination modes of A-C in the table to statistically analyze the residual rate of dormant bodies and the recovery efficiency of internal reference strains after Percoll solution treatment. The recovery rate determination between samples was repeated three times.

[0223] This paper evaluates the effectiveness of the Percoll method from a quantitative perspective.

[0224] The specific operation is as follows:

[0225] In order to realize the accurate counting of spores, 20μl of spores and 100μl of spores were taken, and the combination modes A to C shown in the table were added, and then the samples were placed on the vortex shaker for 10min for mixing, and then the samples were added to the upper part of the 80-40% Percoll solution, and centrifuged at 4℃, 14000x g, 20min. Carefully avoid the precipitate, among them, group A is mainly used to quantitatively evaluate the treatment effect of Percoll strategy on spores, because the two spores can show obvious morphological differences under 60X magnification. Therefore, the cell recovery effect of the two spores after Percoll treatment is counted respectively. In contrast, group B is mainly used to quantitatively evaluate the treatment effect of Percoll strategy on spores. Considering that there is no significant difference between spores, this paper only explains the residual rate of the total amount of spores after treatment. Group C, however, incorporates fluorescently labeled vegetative cells (red / green) into the dormant body for systemically evaluating the residual rate of the dormant body and the recovery efficiency of the vegetative cells after Percoll treatment. The results are shown in Figure 11As shown, according to the test results of different groups of A-C, the residual rate of the mold spore group (A) is relatively low, and is maintained below 0.50%. The cell residual rate of the bacterial spore group (B) is 1.05%. When the complexity of the microbial flora system continues to increase, the removal effect of spores and spores appears to be reduced to a certain extent, but still maintains a residual rate below 10%. At the same time, according to the high recovery rate of the internal reference cells, it can be seen that the method can indeed achieve efficient recovery of the microbial flora and effective enrichment of the dormant bodies to a certain extent.

[0226] Example 6: Summary of experimental effects of the present application

[0227] The summary of the experimental effects of the present application is shown in Table 2.

[0228] Table 2: Summary of experiments

[0229]

[0230]

[0231] Although the present application has been disclosed with the above preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for simultaneous enrichment of both vegetative-resting forms, characterized in that, The method comprises the following steps: (1) adding a cell extraction solution to the sample, mixing and centrifuging to collect the supernatant; adding a cell extraction solution to the precipitate, mixing and centrifuging to collect the supernatant; and combining the two supernatants; (2) centrifuging and filtering the combined supernatant obtained in step (1), high-speed centrifuging the supernatant, discarding the supernatant, resuspending the precipitate obtained after high-speed centrifugation, and obtaining a bacterial suspension; (3) adding the bacterial suspension obtained in step (2) to the upper layer of an iodixanol solution with a concentration of 40-80%, high-speed centrifuging, taking the lower layer solution after centrifugation, adding a buffer solution to the solution and high-speed centrifuging, resuspending the precipitate obtained, and obtaining a resuspension; (4) configuring a double-layer concentration gradient Percoll separation solution: adding a 20-50% concentration Percoll solution to the upper layer containing a 70-90% concentration Percoll to obtain a double-layer concentration gradient Percoll separation solution; (5) adding the resuspension obtained in step (3) to the upper layer of the double-layer concentration gradient Percoll separation solution obtained in step (4), high-speed centrifuging, and obtaining the precipitate, which is the dormant body; (6) adding a sodium chloride solution to the supernatant obtained after centrifugation in step (5), high-speed centrifuging, and taking the precipitate, which is the trophozoite.

2. The method of claim 1, wherein, In step (1): The cell extraction solution is prepared by adding Tween 20 to a system containing a final concentration of 10-50 mM sodium pyrophosphate, so that the mass fraction of Tween 20 is 0.1-1%, and the solvent in the system is a PBS buffer solution with a pH of 7.0-7.8; Preferably, the cell extraction solution is mixed at a mass-volume ratio of 1:1-2; Preferably, the mixing is performed using a vortex shaker under the following conditions: 1500-3500 rpm, 20-45 min; and the centrifugation conditions are 10-50 rpm, 1-2 min.

3. The method according to claim 1 or 2, characterized in that, In step (2): The centrifugation conditions for the combined supernatant are 10-50 rpm, 1-2 min; Preferably, the high-speed centrifugation conditions are 8000-15000 rpm, 2-8 min; Preferably, the precipitate is resuspended using a sterile PBS buffer solution.

4. The method according to any one of claims 1 to 3, characterized in that, In step (3): The high-speed centrifugation conditions after adding the bacterial suspension to the upper layer of the iodixanol solution are 10000-15000 rpm, 20-50 min; Preferably, the conditions for adding an equal amount of PBS buffer solution to the solution and high-speed centrifuging are 8000-12000 rpm, 2-5 min.

5. The method according to any one of claims 1 to 4, characterized in that, In step (4): The Percoll concentration of the upper layer is 40-45%, preferably 40%; and the Percoll concentration of the lower layer is 80-85%, preferably 80%.

6. The method according to any one of claims 1 to 5, characterized in that, In step (5), the high-speed centrifugation conditions are 8000-14000 rpm, 2-10 min.

7. The method according to any one of claims 1 to 6, characterized in that, In step (6): The concentration of sodium chloride is 0.75-0.90%, and the addition amount of sodium chloride is 0.8-1.5 times the volume of the supernatant based on the supernatant. Preferably, the high-speed centrifugation condition is 8000-12000 rpm, 2-10 min.

8. The method according to any one of claims 1 to 7, characterized in that, The sample includes, but is not limited to, Daqu, soil, fermented grains, solid-state fermentation, cheese, cocoa fermentation, sludge, sauce, tea, etc.

9. The method according to any one of claims 1-8, used in the extraction of single cells in complex microbial flora.

10. The method according to any one of claims 1-8, used in a kit for preparing single cells in complex microbial flora or in the separation of vegetative cells and dormant cells, detection of living cells.

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