Microorganism single cell sorting, collecting and culturing method

Abstract

The invention relates to the field of microorganisms, in particular to a microorganism single cell sorting, collecting and culturing method. The LIFT single cell separation technology and the Raman spectrum technology are combined, rapid identification and culture of specific microorganism single cells in a complex sample are achieved, cell damage is reduced, and blind single cell sorting is avoided. The method is easy to operate, the pretreatment process is convenient, and industrial application is facilitated; the sterilized pure water is used as a micro-liquid separation medium, the components are simple, the Raman spectrum is simple and uniform, and downstream microorganism Raman identification, separation and culture are facilitated; the method can be used as samples of intestinal microflora for sorting low-oxygen microorganisms such as saliva, excrement, urine, tongue coating and the like; the method can also be applied to samples of complex microbial communities, such as low-oxygen environments of deep sea, soil, sludge and the like, and sorting culture of unculturable microorganisms; the method can also be applied to screening and application of special functional microorganisms in the industrial fields of fermentation, pharmacy and the like; the method has great market application potential in micro-ecological research.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a method for sorting, collecting and culturing single cells of microorganisms. Background Art

[0002] Traditional biological research on biological cells is mostly based on the level of group cells. This research method usually involves averaging analysis of a large number of cells to obtain information about the overall behavior and function of the cell group. However, this method ignores the heterogeneity between cells, that is, the differences in gene expression, physiological state and response between different cells (Liu Jia. Research and application of single-cell analysis methods based on in vivo microextraction and surface plasmon optical detection [D]. Nanjing University, 2017.). Studies have shown that in cell populations, even between different individual cells with consistent genomic information, their phenotypes have extremely significant differences, including epigenetic regulation, environmental factors and random gene expression (Zhu Shuishan, Wang Zhenjie, Zhang Jixiang. Widespread expression of random monoalleles [J]. Chemistry of Life, 2009.). Although it can provide some macroscopic biological information, research at the level of group cells may in some cases obscure the true state of individual cells, that is, it cannot reveal the complex heterogeneity and individual differences within the cell (Liu Zihui. Research on single-cell elemental analysis based on ICP-MS [D]. Huazhong Agricultural University, 2018.). Therefore, although traditional research methods at the population cell level play an important role in biology, in recent years, with the development of single-cell analysis technology, more and more studies have begun to focus on biological phenomena at the single-cell level in order to more accurately understand the nature and complexity of life activities (An Ning, Zhang Fuyan, Qin Bo. Application of single-cell transcriptome sequencing in age-related macular degeneration research [J]. International Journal of Ophthalmology, 2022.).

[0003] There are many methods for single cell sorting, including Raman spectroscopy, flow cytometry, laser induced forward transfer (LIFT), dilution method, micromanipulation method, membrane diffusion culture method, fluorescence activated cell sorting, magnetic bead sorting, laser microdissection sorting, microfluidic sorting and magnetic cell sorting. The development of single cell sorting technology has greatly promoted the research of life sciences, but it still faces challenges such as detection throughput, cost and data analysis complexity (Xie Jingsheng, Ye Zilu. Single cell proteomics based on mass spectrometry technology [J]. Progress in Biochemistry and Biophysics, 2024.).

[0004] Raman spectroscopy has significant application potential in single-cell separation and analysis. This technology can reveal the chemical composition and molecular structure of cells without labeling and loss, thereby achieving accurate identification and functional phenotype detection of single cells (Wang Xixian, Sun Qing, Diao Zhidian, et al. Progress in the application of Raman spectroscopy in single-cell phenotypic detection and sorting [J]. Synthetic Biology, 2022.). In microbial research, Raman spectroscopy is used to quickly and accurately identify target microorganisms. Combined with single-cell separation technology, single-cell strains can be obtained for subsequent culture and analysis. This method overcomes the shortcomings of traditional culture technology, such as long time consumption and easy failure (Wang Zhenggan, Zhou Zhihui. Application of Raman spectroscopy in microbial research [J]. World Latest Medical Information Abstracts, 2019.), and is an important tool in biotechnology.

[0005] Laser induced forward transfer (LIFT) uses laser pulses to transfer single cells from one location to another, thereby achieving precise cell separation and subsequent cultivation. Some experiments have shown that the average culture recovery rate of Escherichia coli is 58%, while that of Lactobacillus rhamnosus GG (LGG) is only 22% (P. Liang, Huan Wang et al. "Laser induced isolation and cultivation of single microbial cells." bioRxiv (2020).). LIFT technology has certain limitations. Based on LIFT single-cell separation technology, samples need to undergo certain pretreatments. During the pretreatment process, factors such as cell washing conditions and time have different degrees of damage to cells, which affect the survival rate after cell sorting. In addition, the heat and force of the laser are generated during the LIFT sorting process. The laser acts on the sacrificial layer to generate high-temperature steam. The generated heat is absorbed by the cells near the sacrificial layer, resulting in cell damage and death. This greatly reduces the success rate of single-cell live sorting, resulting in a very low probability of live cells being sorted. Although this technology has significant advantages in single cell isolation and culture, its cell survival rate and culture success rate still need to be further optimized.

[0006] The study of microorganisms in hypoxic environments also involves the study of the adaptability of microorganisms to the environment. For example, in the study of marine hypoxic zones, microorganisms have evolved a unique way of breathing in order to adapt to the hypoxic environment, and their community structure and functional activity are significantly different from those in the oxygen-rich environment (Wang Yantao, Chen Quanrui, Ming Hongxia, et al. Research Progress on Microbial Effects in Marine Hypoxic Zones [J]. Marine Environmental Science, 2020.), so special technical means are needed to study these microorganisms. At present, no single-cell pretreatment and sorting culture methods involving hypoxic microorganisms have been found. Summary of the invention

[0007] The present invention combines LIFT single cell separation technology with Raman spectroscopy technology, collects Raman spectra of single cells or spores for identification and separation and culture, and realizes rapid identification and culture of specific microbial single cells in complex samples, reduces cell damage, and avoids blind sorting. Based on this, the present invention is completed.

[0008] In a first aspect, the present invention provides a method for sorting and culturing microbial single cells by combining LIFT single cell sorting with Raman spectroscopy, the method comprising the following steps:

[0009] S1. Sample pretreatment: Take the microbial suspension, wash it, centrifuge it and resuspend it to obtain a reserve solution;

[0010] S2. Raman spectrum acquisition: Take the bacterial solution pre-treated in step S1, place it on the liquid medium of the Raman sorting chip, observe the sample under a Raman microscope, and collect Raman spectra of microorganisms and cells;

[0011] S3. Sorting: positioning the laser point to the cells to be tested in step S2, and performing ejection sorting on the target cells, wherein the medium of the cells to be tested is pure water;

[0012] S4. Receiving: receiving the cells sorted in step S3 with a dedicated receiver;

[0013] S5. Cultivation: The cells received in step S4 are transferred and cultured.

[0014] Furthermore, in the sample pretreatment in step S1, when the microorganism is a hypoxic or anaerobic microorganism, the whole pretreatment is performed in an anaerobic glove box.

[0015] Furthermore, the gases in the anaerobic glove box are hydrogen (5%), nitrogen (90%) and carbon dioxide (5%).

[0016] Furthermore, the volume of the microbial suspension in step S1 is selected from 20-100 μL.

[0017] Furthermore, sterile oxygen-free deionized water is used for cleaning in step S1.

[0018] Furthermore, the centrifugal speed in step S1 is selected from 3000-5000 rpm, preferably 3000 rpm.

[0019] Furthermore, the centrifugation time in step S1 is selected from 3-15 min, preferably 5 min.

[0020] Furthermore, in the step S2 of collecting Raman spectra, when the microorganisms are hypoxic or anaerobic microorganisms, the collection is performed in a closed device.

[0021] Furthermore, nitrogen is introduced into the closed device to create a low oxygen state in the closed device.

[0022] Furthermore, the liquid medium in step S2 is sterile pure water.

[0023] Furthermore, the volume of the sterilized pure water is 1 μL.

[0024] Furthermore, in the Raman spectrum collection in step S2, the wavelength of the Raman laser incident light source is selected from 455-625nm, preferably 532nm.

[0025] Furthermore, in the Raman spectrum acquisition in step S2, the Raman shift scanning range is selected from 400-3600cm -1 .

[0026] Furthermore, in the step S2, the Raman spectrum is collected and the laser intensity of the Raman scan is selected from 1-3mw.

[0027] Furthermore, in the Raman spectrum collection in step S2, the laser scanning time is selected from 1-3s.

[0028] Furthermore, in the step S3, when the microorganisms are hypoxic or anaerobic microorganisms, the sorting is performed in a closed device.

[0029] Furthermore, nitrogen is introduced into the closed device to place the closed device in a low oxygen state.

[0030] Furthermore, in the ejection sorting in step S3, the sorting laser energy is selected from 50-150mJ.

[0031] Furthermore, in the ejection sorting in step S3, the sorting laser energy is selected from 50-80mJ, 80-120mJ and / or 120-150mJ.

[0032] Furthermore, the ejection separation medium in step S3 is sterile pure water.

[0033] Furthermore, when the microorganisms received in step S4 are hypoxic or anaerobic microorganisms, they are received in a closed device.

[0034] Furthermore, nitrogen is introduced into the closed device to place the closed device in a low oxygen state.

[0035] Furthermore, when the microorganisms transferred in step S5 are hypoxic or anaerobic microorganisms, dry ice non-contact operation is adopted.

[0036] Furthermore, sterile water is contained in the operating container, dry ice is placed in the sterile water, and the microbial sample is placed in carbon dioxide smoke.

[0037] Furthermore, in the culturing in step S5, when the microorganisms are hypoxic or anaerobic microorganisms, anaerobic glove box is used for culturing.

[0038] Furthermore, the gases in the anaerobic glove box are hydrogen (5%), nitrogen (90%) and carbon dioxide (5%).

[0039] Further, the microbial single cell includes but is not limited to Bacillus licheniformis, Lactobacillus crispatus, Bifidobacterium longum, Bifidobacterium adolescentis and unculturable microorganisms.

[0040] In one embodiment of the present invention, the microbial single cell is Bacillus licheniformis, and the culture conditions of step S5 are: soy tryptone broth as the basic culture medium; the pH range of the culture medium is selected from: 6.5-7.5; the culture temperature range is selected from: 35-37°C, and the shaking speed is selected from 150-200rpm.

[0041] In one embodiment of the present invention, the microbial single cell is Lactobacillus crispatus, the culture environment of step S5 is an anaerobic glove box, and the gases in the anaerobic glove box are hydrogen, nitrogen and carbon dioxide; the culture conditions are: MRS broth with glycogen added as the basic culture medium, and the concentration of the glycogen is 10g / L; the culture medium is added with mixed trace elements and mixed vitamins, and the trace elements include Nitrilotriacetic acid, MgSO4·7H2O, MnSO4·H2O, NaCl, FeSO4·7H2O, CoCl2·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3 and Na2MoO4·2H2O; the trace element concentration ratio is 150:300:50:100:10:10:10:1:1:1:1; the vitamins include Biotin, Folicacid, Vitamin B12 and Vitamin B1; the concentration ratio of the vitamins is 20:20:1:5; the pH range of the culture medium is selected from: 4.5-6.5; the culture temperature range is selected from: 35-37°C, and the shaking speed is selected from: 150-200rpm.

[0042] In one embodiment of the present invention, the microbial single cell is Bifidobacterium longum, and the culture conditions of step S5 are: using GAM broth as the basic culture medium, without adding a matching antibacterial agent, adding mixed trace elements and mixed vitamins, the trace elements include Nitrilotriacetic acid, MgSO4·7H2O, MnSO4·H2O, NaCl, FeSO4·7H2O, CoCl2·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3 and Na2MoO4·2H2O; the trace element concentration ratio is 150:300:50:100:10:10:10:1:1:1:1; the vitamins include Biotin, Folic acid, Vitamin B12 and Vitamin B1; the concentration ratio of the vitamins is 20:20:1:5; the pH range of the culture medium is selected from: 6.5-7.5, the culture temperature range is selected from: 35-37°C, and the shaking speed is selected from: 150-200rpm.

[0043] In one embodiment of the present invention, the microbial single cell is Bifidobacterium adolescentis, and the culture conditions of step S5 are: using GAM broth as the basic culture medium, without adding a matching antibacterial agent, adding mixed trace elements and mixed vitamins, the trace elements include Nitrilotriacetic acid, MgSO4·7H2O, MnSO4·H2O, NaCl, FeSO4·7H2O, CoCl2·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3 and Na2MoO4·2H2O; the trace element concentration ratio is 150:300:50:100:10:10:10:1:1:1:1; the vitamins include Biotin, Folic acid, Vitamin B12 and Vitamin B1; the concentration ratio of the vitamins is 20:20:1:5; the pH range of the culture medium is selected from: 6.5-7.5, the culture temperature range is selected from: 35-37°C, and the shaking speed is selected from: 150-200rpm.

[0044] Furthermore, sources of the microbial single cell sample include but are not limited to intestinal microbial communities and complex microbial communities.

[0045] Furthermore, sources of intestinal microbial community samples include but are not limited to saliva, feces, urine and tongue coating.

[0046] Furthermore, sources of complex microbial community samples include but are not limited to the deep sea, soil, sludge and other low-oxygen environments.

[0047] In a second aspect, the present invention provides a culture medium for Bacillus licheniformis, which can be used to culture Bacillus licheniformis separated by LIFT single cells and Raman spectroscopy; the method for separation by LIFT single cells and Raman spectroscopy is as shown in the first aspect of the present invention; the culture medium uses soy tryptone broth as a basic culture medium.

[0048] Furthermore, the pH range of the culture medium is selected from: 6.5-7.5.

[0049] In a third aspect, the present invention provides a culture medium for Lactobacillus crispatus, which can be used to culture Lactobacillus crispatus separated by LIFT single cells and Raman spectroscopy; the method for separation by LIFT single cells and Raman spectroscopy is as shown in the first aspect of the present invention; the culture medium uses MRS broth with glycogen as the basic culture medium, and adds mixed trace elements and mixed vitamins.

[0050] Furthermore, the pH range of the culture medium is selected from: 4.5-6.5.

[0051] Furthermore, the glycogen is selected from one or more types of dextran.

[0052] Furthermore, the polysaccharide is selected from animal liver starch, glycogen, bovine liver extract or river mussel extract, and the concentration of the extract is 95%.

[0053] Furthermore, the concentration range of the added glycogen is selected from 5-16 g / L, preferably 10 g / L.

[0054] Furthermore, the trace elements added to the culture medium include Nitrilotriacetic acid, MgSO4·7H2O, MnSO4·H2O, NaCl, FeSO4·7H2O, CoCl2·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3 and Na2MoO4·2H2O.

[0055] Furthermore, the concentration ratio of trace elements added to the culture medium is 150:300:50:100:10:10:10:1:1:1:1.

[0056] Furthermore, the vitamins added to the culture medium specifically include Biotin, Folic acid, Vitamin B12 and Vitamin B1.

[0057] Furthermore, the concentration ratio of the vitamins added to the culture medium is Biotin: Folic acid: Vitamin B12: Vitamin B1=20:20:1:5.

[0058] In a fourth aspect, the present invention provides a culture medium for Bifidobacterium longum, which can be used to culture Bifidobacterium longum separated by LIFT single cells and Raman spectroscopy; the method for separation by LIFT single cells and Raman spectroscopy is as shown in the first aspect of the present invention; the culture medium uses GAM broth as the basic culture medium, does not add a matching antibacterial agent, and adds mixed trace elements and mixed vitamins.

[0059] Furthermore, the pH range of the culture medium is selected from: 6.5-7.5.

[0060] Furthermore, the trace elements added to the culture medium include Nitrilotriacetic acid, MgSO4·7H2O, MnSO4·H2O, NaCl, FeSO4·7H2O, CoCl2·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3 and Na2MoO4·2H2O.

[0061] Furthermore, the concentration ratio of trace elements added to the culture medium is 150:300:50:100:10:10:10:1:1:1:1.

[0062] Furthermore, the vitamins added to the culture medium specifically include Biotin, Folic acid, Vitamin B12 and Vitamin B1.

[0063] Furthermore, the concentration ratio of the vitamins added to the culture medium is Biotin: Folic acid: Vitamin B12: Vitamin B1=20:20:1:5.

[0064] In a fifth aspect, the present invention provides a culture medium for Bifidobacterium adolescentis, which can be used to culture Bifidobacterium longum separated by LIFT single cell and Raman spectroscopy; the method for separation by LIFT single cell and Raman spectroscopy is as shown in the first aspect of the present invention; the culture medium uses GAM broth as the basic culture medium, does not add a matching antibacterial agent, and adds mixed trace elements and mixed vitamins.

[0065] Furthermore, the pH range of the culture medium is selected from: 6.5-7.5.

[0066] Furthermore, the trace elements added to the culture medium include Nitrilotriacetic acid, MgSO4·7H2O, MnSO4·H2O, NaCl, FeSO4·7H2O, CoCl2·6H2O, CaCl2, ZnSO4·7H2O, CuSO4·5H2O, H3BO3 and Na2MoO4·2H2O.

[0067] Furthermore, the concentration ratio of trace elements added to the culture medium is 150:300:50:100:10:10:10:1:1:1:1.

[0068] Furthermore, the vitamins added to the culture medium specifically include Biotin, Folic acid, Vitamin B12 and Vitamin B1.

[0069] Furthermore, the concentration ratio of the vitamins added to the culture medium is Biotin: Folic acid: Vitamin B12: Vitamin B1=20:20:1:5.

[0070] In a sixth aspect, the present invention provides an application of a microbial sorting and culturing method combining LIFT single cell sorting with Raman spectroscopy in microbial fermentation, wherein the method is as described in the first aspect of the present invention.

[0071] Furthermore, sources of the microbial single cell sample include but are not limited to intestinal microbial communities and complex microbial communities.

[0072] Furthermore, sources of intestinal microbial community samples include but are not limited to saliva, feces, urine and tongue coating.

[0073] Furthermore, sources of complex microbial community samples include but are not limited to the deep sea, soil, sludge and other low-oxygen environments.

[0074] In the seventh aspect, the present invention provides an application of a microbial sorting and cultivation method combining LIFT single-cell sorting with Raman spectroscopy in microbial pharmaceutical manufacturing, wherein the method is as described in the first aspect of the present invention; the pharmaceutical manufacturing includes the screening and cultivation of functional microorganisms.

[0075] Furthermore, sources of the microbial single cell sample include but are not limited to intestinal microbial communities and complex microbial communities.

[0076] Furthermore, sources of intestinal microbial community samples include but are not limited to saliva, feces, urine and tongue coating.

[0077] Furthermore, sources of complex microbial community samples include but are not limited to the deep sea, soil, sludge and other low-oxygen environments.

[0078] Beneficial Effects

[0079] The present invention has the following advantages:

[0080] (1) The present invention pre-treats hypoxia and anaerobic samples, and after sorting single cells under ventilation, transfers the samples to an anaerobic glove box on dry ice for continued culture, thereby reducing cell damage and improving single cell survival rate;

[0081] (2) The present invention is simple to operate and has a convenient pre-treatment process, which avoids the problem of high cost of transformation and upgrading, and is more conducive to industrial application;

[0082] (3) Using sterilized pure water as the microfluid sorting medium, the water has a simple composition and a simple and uniform Raman spectrum, which is beneficial to the downstream Raman identification, separation and culture of microorganisms; it can achieve rapid Raman identification and post-culture of microorganisms in complex samples, avoiding blind single-cell sorting;

[0083] (4) It can be applied to the sorting of low-oxygen microorganisms in samples of intestinal microbial communities such as saliva, feces, urine, tongue coating, etc.; it can also be applied to the sorting and cultivation of microorganisms that cannot be cultured in samples of complex microbial communities, such as low-oxygen environments such as the deep sea, soil, sludge, etc.; it has great market application potential in microecological research;

[0084] (5) It can also be used to screen special functional microorganisms in industrial fields such as fermentation and pharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 It is the sample preparation and single cell sorting culture process.

[0086] Figure 2 Microscopic images of the survival rate and cell damage of Bacillus licheniformis at different centrifugation rates.

[0087] Figure 3 The survival rate of Lactobacillus culture in different sorting modes.

[0088] Figure 4 Images of strains sorted from single cells for each sample;

[0089] Note: Figure 4 A is Lactobacillus crispatus Lc-Q1; Figure 4 B is Lactobacillus crispatus Lc-MV6-4; Figure 4 C is Bifidobacterium longum.

[0090] Figure 5 The survival rate of single-cell Raman spectroscopy acquisition and sorting combined with culture of Lactobacillus crispatus and Bifidobacterium longum was investigated.

[0091] Figure 6The survival rate and screening accuracy of single-cell Raman spectroscopy combined with sorting of Bifidobacterium adolescentis in fecal samples. DETAILED DESCRIPTION

[0092] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0093] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0094] Example 1 Sorting culture (centrifugation condition screening)

[0095] Bacillus licheniformis, Lactobacillus crispatus, Bifidobacterium longum and Bifidobacterium adolescentis were sorted and cultured respectively; anaerobic and hypoxic microorganisms were handled in an anaerobic glove box throughout the process to ensure a low oxygen environment.

[0096] A. Sample Preparation

[0097] (1) taking activated bacterial liquids of Bacillus licheniformis, Lactobacillus crispatus, Bifidobacterium longum and Bifidobacterium adolescentis respectively, adding them into sterile deionized water, and washing the bacterial cells;

[0098] (2) Centrifuge the bacterial suspension in (1) separately and discard the supernatant;

[0099] (3) Add sterile deionized water to resuspend, mix well, and set aside;

[0100] There are four centrifugation conditions in setting step (2): rotation speed: 3000 rpm, centrifugation for 5 min; rotation speed: 5000 rpm, centrifugation for 5 min; rotation speed: 8000 rpm, centrifugation for 2 min; rotation speed: 8000 rpm, centrifugation for 5 min; experiments were carried out separately, and the morphology of the test bacteria under different centrifugation conditions was observed under a microscope.

[0101] B. Single Cell Sorting

[0102] (1) Pipette the pre-treated middle layer bacterial solution, spot it on the Raman sorting chip, air-dry it and then load it into the machine;

[0103] (2) Observe the sample in (1) under a Raman microscope;

[0104] (3) Positioning the laser spot on the cell to be tested;

[0105] (4) The target cells are ejected and sorted, and the sorting laser energy is 50mJ-80mJ, depending on the cell size.

[0106] (5) The sorted cells are received in a dedicated receiver containing 30 μl of culture medium.

[0107] C. Single cell culture

[0108] (1) Transferring the single cells received by the receiver to a culture medium compatible with the bacteria;

[0109] (2) Place the culture tube in (2) in a shaker at 35-37°C and 150-200 rpm;

[0110] (3) After 1-5 days of culture, observe the turbidity of the culture medium in the culture tube and calculate the survival rate.

[0111] When the isolated bacteria are Bacillus licheniformis, the bacteria are transferred to a 5 ml culture tube containing 1 ml of soy tryptone broth in step (1) for subsequent operations.

[0112] When the separated bacteria are Lactobacillus crispatus, the culture medium in step (1) is based on MRS broth, with glycogen (10 g / L) as supplementary nutrition, and mixed trace elements and mixed vitamins are added, and the trace elements are specifically 15 mg / L of Nitrilotriacetic acid, 30 mg / L of MgSO4·7H2O, 5 mg / L of MnSO4·H2O, 10 mg / L of NaCl, 1 mg / L of FeSO4·7H2O, 1 mg / L of CoCl2·6H2O, 1 mg / L of CaCl2, 1 mg / L of ZnSO4·7H2O, 0.1 mg / L of CuSO4·5H2O, 0.1 mg / L of H3BO3, and 0.1 mg / L of Na2MoO4·2H2O. The specific vitamins are Biotin 2mg / L, Folic acid 2mg / L, Vitamin B120.1mg / L, Vitamin B15mg / L.

[0113] When the selected bacteria are Bifidobacterium longum, the culture medium in step (1) is a modified GAM broth-based culture medium, without adding a matching antibacterial agent, and adding mixed trace elements and mixed vitamins. The trace elements are specifically Nitrilotriacetic acid 15mg / L, MgSO4·7H2O 30mg / L, MnSO4·H2O 5mg / L, NaCl 10mg / L, FeSO4·7H2O 1mg / L, CoCl2·6H2O 1mg / L, CaCl2 1mg / L, ZnSO4·7H2O 1mg / L, CuSO4·5H2O0.1mg / L, H3BO30.1mg / L, Na2MoO4·2H2O 0.1mg / L. The vitamins are specifically Biotin 2mg / L, Folic acid 2mg / L, Vitamin B120.1mg / L, Vitamin B15mg / L.

[0114] When the selected bacteria are adolescent bifidobacterium, the culture medium in step (1) is a modified GAM broth-based culture medium, without adding a matching antibacterial agent, and adding mixed trace elements and mixed vitamins. The trace elements are specifically Nitrilotriacetic acid 15mg / L, MgSO4·7H2O 30mg / L, MnSO4·H2O 5mg / L, NaCl 10mg / L, FeSO4·7H2O 1mg / L, CoCl2·6H2O 1mg / L, CaCl2 1mg / L, ZnSO4·7H2O 1mg / L, CuSO4·5H2O0.1mg / L, H3BO30.1mg / L, Na2MoO4·2H2O 0.1mg / L. The vitamins are specifically Biotin 2mg / L, Folic acid 2mg / L, Vitamin B120.1mg / L, Vitamin B15mg / L.

[0115] D. Test results

[0116] like Figure 2 As shown in the figure, under the microscope, the Bacillus licheniformis cells were damaged to different degrees under different centrifugation conditions. The cells were in better condition when centrifuged at 3000 rpm for 5 minutes than those under other conditions.

[0117] When Bacillus licheniformis was centrifuged at 3000 rpm for 5 min, 60 single cells were sorted and 12 tubes of turbid bacterial liquid were grown, with a survival rate of 20%;

[0118] When Bacillus licheniformis was centrifuged at 5000 rpm for 5 min, 120 single cells were sorted and three tubes of turbid bacterial liquid were grown, with a survival rate of 2.5%;

[0119] When the centrifugation condition of Bacillus licheniformis was 8000 rpm for 2 min, 96 single cells were sorted, 0 tubes of turbid bacterial liquid grew out, and the survival rate was 0%.

[0120] When the centrifugation condition was 8000 rpm for 5 min, 0 tubes of turbid bacterial liquid grew out of Bacillus licheniformis, and the survival rate was 0%.

[0121] Similarly, under the microscope, different centrifugation conditions showed different degrees of damage to the cells of Lactobacillus crispatus, Bifidobacterium longum and Bifidobacterium adolescentis. The cells were better when centrifuged at 3000 rpm for 5 minutes than under other conditions. When the centrifugation speed reached 8000 rpm, the cell survival rate was 0% after a period of centrifugation.

[0122] When the rotation speed is too low, single bacterial cells cannot be separated, resulting in poor separation effect, which affects subsequent operations.

[0123] Therefore, the centrifugation condition of 3000 rpm for 5 min was selected for subsequent experiments.

[0124] Example 2 Cultivation in different sorting modes (air-dried and solution medium)

[0125] The facultative anaerobic lactobacillus single-cell Lactobacillus crispatus Lc-Q1 (Lactobacillus crispatus) was sorted and cultured.

[0126] A. Sample Preparation

[0127] (1) The whole process is carried out in an anaerobic glove box;

[0128] (2) taking activated lactobacillus liquid, adding it into sterile deionized water, and washing the bacteria;

[0129] (3) Centrifuge the bacterial solution in (1) at 3000 rpm for 5 min and discard the supernatant;

[0130] (4) Add sterile oxygen-free deionized water to resuspend, mix well, and set aside;

[0131] B. Single cell sorting (air drying)

[0132] (1) The whole process is carried out in a closed device with nitrogen gas;

[0133] (2) Absorb the pre-treated middle layer bacterial solution and spot it on the Raman sorting chip;

[0134] (3) After spotting, air dry in the fume hood before loading onto the machine;

[0135] (4) Position the laser spot on the cells to be tested and perform ejection sorting on the target cells. The sorting laser energy is 50mJ-80mJ.

[0136] C. Single cell sorting (water medium)

[0137] (1) The whole process is carried out in a closed device with nitrogen gas;

[0138] (2) Absorb the pre-treated middle layer bacterial solution and spot it on the Raman sorting chip;

[0139] (3) Use sterile water as the medium and put it on the machine;

[0140] (4) After spotting, the samples are directly put into the machine for rapid sorting (while the samples are not dry). The sorting laser energy is 80mJ-120mJ.

[0141] D. Single cell culture

[0142] (1) The sorted cells are received in a dedicated receiver (containing 30 μl of anaerobic culture medium);

[0143] (2) The receiver receiving the cells in (1) is immediately transferred to an anaerobic glove box with dry ice, and sterile water is placed inside the operating container, dry ice is placed in the sterile water, and the receiver receiving the cells is placed in carbon dioxide smoke;

[0144] (3) transferring the sorted cells to the packaged anaerobic culture medium for culture;

[0145] (4) After 1-5 days of culture, observe the turbidity of the culture medium in the culture tube and calculate the survival rate.

[0146] The culture medium is based on MRS broth, supplemented with glycogen (10 g / L), mixed trace elements and mixed vitamins, and the trace elements are specifically Nitrilotriacetic acid 15 mg / L, MgSO4·7H2O 30 mg / L, MnSO4·H2O 5 mg / L, NaCl 10 mg / L, FeSO4·7H2O 1 mg / L, CoCl2·6H2O 1 mg / L, CaCl2 1 mg / L, ZnSO4·7H2O 1 mg / L, CuSO4·5H2O 0.1 mg / L, H3BO3 0.1 mg / L, and Na2MoO4·2H2O 0.1 mg / L. The vitamins are specifically Biotin 2 mg / L, Folic acid 2 mg / L, Vitamin B12 0.1 mg / L, and Vitamin B1 5 mg / L. The pH is 4.5, the temperature is 35-37°C, and the shaking speed is 150-200 rpm. The culture is carried out in an anaerobic glove box with a hydrogen concentration of 4%, a carbon dioxide concentration of 5%, and an oxygen concentration of 0 PPM.

[0147] E. Test results

[0148] like Figure 3 As shown, when Lactobacillus crispatus Lc-Q1 was cultured in water medium, 30 single cells of Lactobacillus crispatus were sorted, and 12 tubes of turbid bacterial liquid were grown, with a survival rate of 40%; subsequent collection, sorting and culture can be carried out.

[0149] In the case of air-dried sorting and culture, 60 single cells of Lactobacillus crispatus Lc-Q1 were sorted, and 0 tubes of turbid bacterial liquid grew out, with a survival rate of 0%. Subsequent collection, sorting and culture were not possible.

[0150] When selecting the target strain, the cell survival rate increased after the aqueous solution medium was selected. Therefore, the aqueous solution medium was selected for subsequent experiments.

[0151] Example 3: Raman spectroscopy acquisition and sorting combined with cultivation of facultative anaerobic lactobacillus single cells

[0152] Two strains of Lactobacillus crispatus were sorted and cultured: Lactobacillus crispatus Lc-Q1 (Lactobacillus crispatus) and Lactobacillus crispatus Lc-MV6-4 (Lactobacillus crispatus).

[0153] A. Sample Preparation

[0154] (1) The whole process is carried out in an anaerobic glove box;

[0155] (2) taking activated lactobacillus liquid, adding it into sterile deionized water, and washing the bacteria;

[0156] (3) Centrifuge the bacterial solution in (1) at 3000 rpm for 5 min and discard the supernatant;

[0157] (4) Add sterile oxygen-free deionized water to resuspend, mix well, and set aside;

[0158] B. Single cell collection and sorting (aqueous medium)

[0159] (1) The whole process is carried out in a closed device, and nitrogen is introduced to keep the environment in a low oxygen state;

[0160] (2) Absorb the pre-treated middle layer bacterial solution and spot it on the Raman sorting chip;

[0161] (3) Using sterile water as the medium, spot it on the Raman sorting chip in (2);

[0162] (4) After spotting, the sample was directly put on the machine for Raman acquisition. The wavelength of the Raman laser incident light source was 532nm, and a 600g / mm grating was used to scan 400-3600cm -1 Raman shift within the range, the scanning laser intensity is 1mw, and the laser scanning time is 1s;

[0163] (5) Rapidly sort the cells whose Raman spectra were collected in (4) (during the period when the samples are not dry), and the sorting laser energy is

[0164] 80mJ-120mJ.

[0165] C. Single cell culture

[0166] (1) The sorted cells are received in a dedicated receiver (containing 30 μl of anaerobic culture medium);

[0167] (2) The receiver receiving the cells in (1) is immediately transferred to an anaerobic glove box with dry ice, and sterile water is placed inside the operating container, dry ice is placed in the sterile water, and the receiver receiving the cells is placed in carbon dioxide smoke;

[0168] (3) transferring the sorted cells to the prepared anaerobic culture medium for culture;

[0169] (4) After 1-5 days of culture, observe the turbidity of the culture medium in the culture tube and calculate the survival rate.

[0170] The culture medium is based on MRS broth, with river mussel extract (10g / L) as supplementary nutrition, mixed trace elements and mixed vitamins, the trace elements are specifically Nitrilotriacetic acid 15mg / L, MgSO4·7H2O30mg / L, MnSO4·H2O 5mg / L, NaCl 10mg / L, FeSO4·7H2O 1mg / L, CoCl2·6H2O 1mg / L, CaCl21mg / L, ZnSO4·7H2O 1mg / L, CuSO4·5H2O 0.1mg / L, H3BO30.1mg / L, Na2MoO4·2H2O0.1mg / L. The vitamins are specifically Biotin 2mg / L, Folic acid 2mg / L, Vitamin B120.1mg / L, VitaminB15mg / L. The pH is 4.5, the temperature is 35-37°C, and the shaking speed is 150-200 rpm. The culture is carried out in an anaerobic glove box with a hydrogen concentration of 4%, a carbon dioxide concentration of 5%, and an oxygen concentration of 0 PPM.

[0171] D. Test results

[0172] like Figure 4 As shown in A, it is a picture of the Lactobacillus crispatus Lc-Q1 strain obtained by water medium sorting; Figure 4 Shown in B is a picture of the Lactobacillus crispatus Lc-MV6-4 strain obtained by water medium sorting.

[0173] Under air-dried medium conditions, the survival rate of the strain was 0%, and subsequent sorting, collection and cultivation work could not be carried out. However, under aqueous medium conditions, the strain survived and could be sorted, collected and cultivated by selecting appropriate laser energy according to the molecular size.

[0174] like Figure 5 As shown, when Lactobacillus crispatus Lc-Q1 was sorted and cultured after Raman spectrum acquisition, 60 single cells of Lactobacillus crispatus Lc-Q1 were sorted, and 11 tubes of turbid bacterial liquid were grown, with a survival rate of 18.33%.

[0175] After the Raman spectrum of Lactobacillus crispatus Lc-MV6-4 was collected, 20 single cells of Lactobacillus crispatus Lc-MV6-4 were sorted and cultured, and 5 tubes of turbid bacterial liquid were grown, with a survival rate of 25%.

[0176] Example 4: Raman spectrum collection and sorting of single cells of completely anaerobic Bifidobacterium for cultivation

[0177] Bifidobacterium longum was sorted and cultured.

[0178] A. Sample Preparation

[0179] (1) The whole process is carried out in an anaerobic glove box;

[0180] (2) taking activated lactobacillus liquid, adding it into sterile deionized water, and washing the bacteria;

[0181] (3) Centrifuge the bacterial solution in (1) at 3000 rpm for 5 min and discard the supernatant;

[0182] (4) Add sterile oxygen-free deionized water to resuspend, mix well, and set aside;

[0183] B. Single cell sorting (air drying)

[0184] (1) The whole process is carried out in a closed device with nitrogen gas;

[0185] (2) Absorb the pre-treated middle layer bacterial solution and spot it on the Raman sorting chip;

[0186] (3) After spotting, air dry in the fume hood before loading onto the machine;

[0187] (4) Position the laser spot on the cells to be tested and perform ejection sorting on the target cells. The sorting laser energy is 50mJ-80mJ.

[0188] C. Single cell collection and sorting (water medium)

[0189] (1) The whole process is carried out in a closed device, and nitrogen is introduced to keep the environment in a low oxygen state;

[0190] (2) Absorb the pre-treated middle layer bacterial solution and spot it on the Raman sorting chip;

[0191] (3) Using sterile water as the medium, spot it on the Raman sorting chip in (2);

[0192] (4) After spotting, the Raman data were collected directly on the machine. The wavelength of the Raman laser incident light source was 532 nm, and a 600 g / mm grating was used, scanning 400-3600 cm -1 Raman shift within the range, the scanning laser intensity is 1mw, and the laser scanning time is 1s;

[0193] (5) Rapidly sort the cells whose Raman spectra were collected in (4) (during the period when the samples are not dry), and the sorting laser energy is

[0194] 80mJ-120mJ.

[0195] D. Single cell culture

[0196] (1) The sorted cells are received in a dedicated receiver (containing 30 μL of anaerobic culture medium);

[0197] (2) The receiver receiving the cells in (1) is immediately transferred to an anaerobic glove box with dry ice, and sterile water is placed inside the operating container, dry ice is placed in the sterile water, and the receiver receiving the cells is placed in carbon dioxide smoke;

[0198] (3) transferring the sorted cells to the packaged anaerobic culture medium for culture;

[0199] (4) After 1-5 days of culture, observe the turbidity of the culture medium in the culture tube and calculate the survival rate.

[0200] The modified GAM broth is used as the basic culture medium, without adding the supporting antibacterial agent, and mixed trace elements and mixed vitamins are added, the specific content is the same as that of lactobacillus culture, pH is: 6.5, temperature is: 35-37℃, and the shaking speed is 150-200rpm. Cultured in an anaerobic glove box, the hydrogen concentration is 4%, the carbon dioxide concentration is 5%, and the nitrogen concentration is 90%.

[0201] E. Test results

[0202] like Figure 4 C shows a picture of the Bifidobacterium longum WF6-11 strain obtained by water medium sorting.

[0203] Under air-dried medium conditions, the survival rate of the strain was 0%, and subsequent sorting, collection and cultivation work could not be carried out. However, under aqueous medium conditions, the strain survived and could be sorted, collected and cultivated by selecting appropriate laser energy according to the molecular size.

[0204] like Figure 5 As shown, when Bifidobacterium longumWF6-11 was sorted and cultured after Raman spectrum acquisition, 40 Bifidobacterium longum single cells were sorted, and 8 tubes of turbid bacterial liquid were grown, with a survival rate of 20%.

[0205] Example 5 Raman spectroscopy acquisition and sorting of single-cell Bifidobacterium adolescentis in fecal samples combined with culture (different media)

[0206] A. Sample Preparation

[0207] (1) The whole process is carried out in an anaerobic glove box;

[0208] (2) Take a fresh stool sample from a healthy person and add it to a 50 ml sterile centrifuge tube filled with nitrogen;

[0209] (3) Weigh 1 g of the sample in (2) into a 15 ml sterile centrifuge tube, add 5 ml of sterile PBS, and vortex for 5 min;

[0210] (4) Filter through a 40 μm cell strainer to obtain 1 ml of filtrate;

[0211] (5) Take the bacterial solution from (4), centrifuge at 3000 rpm for 5 min, and discard the supernatant;

[0212] (6) Add sterile oxygen-free deionized water to resuspend, mix well, and set aside;

[0213] B. Single cell sorting (air drying)

[0214] (5) The whole process is carried out in a closed device with nitrogen gas;

[0215] (6) Absorb the pre-treated middle layer bacterial solution and spot it on the Raman sorting chip;

[0216] (7) After spotting, air dry in the fume hood before loading onto the machine;

[0217] (8) Position the laser spot on the cells to be tested and perform ejection sorting on the target cells. The sorting laser energy is 50mJ-80mJ.

[0218] C. Single cell collection and sorting (sterile water medium)

[0219] (1) The whole process is carried out in a closed device, and nitrogen is introduced to keep the environment in a low oxygen state;

[0220] (2) Absorb the pre-treated middle bacterial liquid, use 1 μL of sterile water as the medium, and spot it on the Raman sorting chip;

[0221] (3) After spotting, the Raman spectrum of the rod-shaped cells was collected directly on the machine. The wavelength of the Raman laser incident light source was 532nm, and a 600g / mm grating was used to scan 400-3600cm -1 Raman shift within the range, the scanning laser intensity is 1mW,

[0222] The laser scanning time is 1s;

[0223] (4) Raman spectrum comparison: cells with a Raman spectrum similarity of more than 90% with that of Bifidobacterium in the Raman database are rapidly sorted (during the sample drying period), and the sorting laser energy is 80mJ-120mJ.

[0224] D. Single cell collection and sorting (trehalose solution medium)

[0225] (1) The whole process is carried out in a closed device, and nitrogen is introduced to keep the environment in a low oxygen state;

[0226] (2) Absorb the pre-treated middle bacterial liquid, use 1-2 μL of trehalose solution as a medium, and spot it on the Raman sorting chip;

[0227] (3) After spotting, the Raman spectrum of the rod-shaped cells was collected directly on the machine. The wavelength of the Raman laser incident light source was 532nm, and a 600g / mm grating was used to scan 400-3600cm -1 Raman shift within the range, the scanning laser intensity is 1mW,

[0228] The laser scanning time is 1s;

[0229] (4) Raman spectrum comparison: cells with a Raman spectrum similarity of more than 90% with that of Bifidobacterium in the Raman database are rapidly sorted (during the sample drying period), and the sorting laser energy is 120mJ-150mJ.

[0230] E. Single cell culture

[0231] (1) The sorted cells are received in a dedicated receiver (containing 30 μL of anaerobic culture medium);

[0232] (2) The receiver receiving the cells in (1) is immediately transferred to an anaerobic glove box with dry ice, and sterile water is placed inside the operating container, dry ice is placed in the sterile water, and the receiver receiving the cells is placed in carbon dioxide smoke;

[0233] (3) transferring the sorted cells to the packaged anaerobic culture medium for culture;

[0234] (4) After 1-5 days of culture, observe the turbidity of the culture medium in the culture tube and calculate the survival rate.

[0235] The modified GAM broth is used as the basic culture medium, without adding the supporting antibacterial agent, and mixed trace elements and mixed vitamins are added, the specific content is the same as that of lactobacillus culture, pH is: 6.5, temperature is: 35-37℃, and the shaking speed is 150-200rpm. Cultured in an anaerobic glove box, the hydrogen concentration is 4%, the carbon dioxide concentration is 5%, and the oxygen concentration is 0PPM.

[0236] F. Test Results

[0237] Fecal microbial suspension samples have high species diversity, and bacteria of different forms or different species of the same form are rich. In such a complex biological environment, when screening single cells of a species, all of them must be picked, cultured, and identified. The combination of single-cell Raman spectroscopy acquisition and sorting can instantly identify and sort out the specified strains for precise culture.

[0238] When the target strain is Bifidobacterium adolescentis, LIFT sorting alone cannot directly obtain the target strain. All the sorted single cells need to be picked and cultured, and the species is identified based on the cultured strains. This process is time-consuming and laborious, and data results cannot be obtained immediately, especially for low-oxygen microorganisms. The operation is complicated and the environmental conditions are harsh.

[0239] When the target strain is Bifidobacterium adolescentis, the Raman spectrum acquisition and sorting can be combined to directly sort the target strain by comparing the acquired spectra, and then the target strain can be specifically cultured for subsequent tests. This process is simple, fast, cost-saving and efficient.

[0240] When single-cell Raman spectroscopy acquisition and sorting are combined, under air-dried medium conditions, the survival rate of the strain is 0%, and subsequent sorting, collection and cultivation work cannot be carried out. However, under solution medium conditions, the appropriate laser energy is selected according to the molecular size, and the strain survives and can be sorted, collected and cultured.

[0241] When single-cell Raman spectroscopy acquisition and sorting are combined, algae polysaccharide solution is selected as the solution medium. There are many interferences in the subsequent bioinformatics analysis after Raman acquisition, and the analysis process is complicated. It is impossible to instantly compare the acquired spectrum with the collected single cells of the target strain, which affects the acquisition accuracy.

[0242] When single-cell Raman spectroscopy acquisition and sorting are combined, a sterile aqueous solution is selected as the solution medium. The Raman acquisition and subsequent bioinformatics analysis process is simple and rapid, and the collected spectrum can be instantly compared with the single cell of the target strain for similarity with high accuracy.

[0243] like Figure 6 As shown, after the fecal microbial suspension sample was collected by Raman spectrum, single cell sorting and culture were performed. 50 rod-shaped single cells with a Raman spectrum similarity of more than 90% with Bifidobacterium adolescentis were sorted, and 14 tubes of turbid bacterial liquid were grown, with a survival rate of 28%. Through single bacterial genome determination, 9 strains were molecularly identified as Bifidobacterium adolescentis with an accuracy of 64%.

Claims

1. A method for sorting and culturing microbial single cells by combining LIFT single cell sorting with Raman spectroscopy, the method comprising the following steps: S1. Sample pretreatment: Take the microbial suspension, wash it, centrifuge it and resuspend it to obtain a reserve solution; S2. Raman spectrum acquisition: Take the bacterial solution pre-treated in step S1, place it on the liquid medium of the Raman sorting chip, observe the sample under a Raman microscope, and collect Raman spectra of microorganisms and cells; S3. Sorting: positioning the laser point to the cells to be tested in step S2, and performing ejection sorting on the target cells, wherein the medium of the cells to be tested is pure water; S4. Receiving: receiving the cells sorted in step S3 with a dedicated receiver; S5. Cultivation: The cells received in step S4 are transferred and cultured.

2. According to the method for sorting and culturing microbial single cells by combining LIFT single cell sorting with Raman spectroscopy as described in claim 1, in the sample pretreatment in step S1, when the microorganism is a hypoxic or anaerobic microorganism, the pretreatment is performed entirely in an anaerobic glove box; the gas in the anaerobic glove box is hydrogen (5%), nitrogen (90%) and carbon dioxide (5%).

3. The method for sorting and culturing microbial single cells by combining LIFT single cell sorting and Raman spectroscopy as claimed in claim 1, wherein the centrifugal speed in step S1 is 3000 rpm and the centrifugation time is 5 min.

4. The method for sorting and culturing microbial single cells by combining LIFT single cell sorting with Raman spectroscopy as claimed in claim 1, wherein the liquid medium in step S2 is sterile pure water.

5. The method for sorting and culturing microbial single cells by combining LIFT single cell sorting and Raman spectroscopy as claimed in claim 1, wherein the wavelength of the Raman laser incident light source for Raman spectrum collection in step S2 is 455-625nm, and the Raman shift scanning range during Raman spectrum collection is 400-3600cm -1 , the laser intensity of Raman scanning is 1-3mw, and the laser scanning time is 1-3s.

6. The method for sorting and culturing microbial single cells by combining LIFT single cell sorting with Raman spectroscopy as claimed in claim 1, wherein the ejection sorting in step S3 uses a sorting laser energy of 50-150 mJ.

7. Application of a microbial sorting and cultivation method combining LIFT single cell sorting and Raman spectroscopy in microbial fermentation, the method as described in claim 1.

8. Application of a microbial sorting and culturing method combining LIFT single cell sorting and Raman spectroscopy in microbial pharmaceutical manufacturing, the method being as described in claim 1; the pharmaceutical manufacturing comprising the screening and culturing of functional microorganisms.

9. The method and application of LIFT single cell sorting combined with Raman spectroscopy for sorting and culturing microbial single cells as described in any one of claims 1 to 8, wherein the microbial single cells include but are not limited to Bacillus licheniformis, Lactobacillus crispatus, Bifidobacterium longum, Bifidobacterium adolescentis and unculturable microorganisms.

10. The method and application of LIFT single cell sorting combined with Raman spectroscopy for sorting and culturing microbial single cells according to any one of claims 1 to 8, wherein the source of the microbial single cell sample includes but is not limited to intestinal microbial communities and complex microbial communities, wherein: The sources of the intestinal microbial community samples include but are not limited to saliva, feces, urine and tongue coating; the sources of the complex microbial community samples include but are not limited to the deep sea, soil, sludge and other low-oxygen environments.

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