Method for high-throughput screening of high-activity lactic acid bacteria based on multi-index fusion and application

Abstract

The invention discloses a method for high-throughput screening of high-activity lactic acid bacteria based on multi-index fusion and application, and belongs to the field of lactic acid bacteria screening, the method comprises the steps of bacterial cell enrichment, single-cell suspension preparation, physiological status and activity evaluation of cells, flow cytometry analysis, cell sorting and strain identification; in the step of enrichment of the bacterial cells, a sample is activated and enriched by using an improved liquid culture medium and an improved solid culture medium, and activated and enriched bacterial suspension is obtained. The invention provides the method for detecting the activity of the fermentation product and the lactic acid bacteria stored for a long time, the method is convenient and rapid, the screening time is greatly shortened, and the screening efficiency is improved.

Description

Technical Field

[0001] This invention belongs to the field of lactic acid bacteria screening, specifically relating to a method and application for high-throughput screening of highly active lactic acid bacteria based on multi-index fusion. Background Technology

[0002] Lactic acid bacteria (LAB) are a class of probiotics widely found in nature, possessing important physiological functions, including regulating intestinal flora balance, enhancing immunity, and inhibiting the growth of pathogenic bacteria. In the food industry, lactic acid bacteria are widely used in fermented dairy products, fermented vegetables, and meat products; in the pharmaceutical field, they are used to develop probiotic preparations for treating intestinal diseases and improving immunity. Therefore, efficient screening of live lactic acid bacteria is of great significance for the development of related industries.

[0003] Traditional screening methods primarily rely on plate culture and biochemical identification, which are limited by time-consuming and labor-intensive processes, insufficient accuracy, and inadequate high-throughput capabilities, failing to meet the demands of modern industry for high-throughput screening. High-throughput screening plays a crucial role in microbial research. It transforms the traditional method of analyzing small numbers of microorganisms one by one, enabling rapid screening of thousands of microorganisms in a short time, significantly accelerating the microbial discovery process. Flow cytometry (FCM) is a high-throughput cell analysis technique capable of performing multi-parameter analysis on large numbers of cells in a short time. In recent years, flow cytometry has been increasingly applied in microbiological research, particularly demonstrating great potential in bacterial activity assessment and screening.

[0004] Currently, high-throughput screening methods for highly active lactic acid bacteria mainly include nucleic acid double staining, cell membrane integrity assessment, cell metabolic activity assessment, cell membrane lipid peroxidation assessment, lactate detection, and extracellular polysaccharide detection. Although each method has its own focus, a single assessment cannot accurately reflect the "high activity" of lactic acid bacteria. For example, nucleic acid double staining / cell membrane integrity assessment mainly reflects the integrity of the cell's physical barrier, but cannot reveal the metabolic activity inside the cell; cell metabolic activity assessment mainly reflects the cell's immediate metabolic capacity, but cannot distinguish between transient stress response and sustainable physiological activity; cell membrane lipid peroxidation assessment is an important indicator of oxidative stress damage, but a single peroxidation level cannot directly indicate whether the cell has died or completely lost its function; while lactate detection is direct evidence of functional activity, but the yield at a single time point cannot represent the long-term viability and stability of the strain, nor can it reflect the cell's own health status; extracellular polysaccharide detection reflects more the strain's stress response or protective mechanism, and cannot directly indicate the strain's survival or metabolic indicators. Summary of the Invention

[0005] Based on the above background technology, the present invention provides a method and application for high-throughput screening of highly active lactic acid bacteria based on multi-index fusion. The method of the present invention is convenient and fast, greatly shortens the screening time, and improves the screening efficiency.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for high-throughput screening of highly active lactic acid bacteria based on multi-index fusion includes: bacterial cell enrichment, preparation of single-cell suspension, assessment of cell physiological state and activity, flow cytometry analysis, cell sorting and bacterial species identification. The bacterial cell enrichment was achieved by activating and enriching the sample using modified liquid culture medium and modified solid culture medium to obtain an activated and enriched bacterial suspension. The bacterial cells are enriched, and the sample is a fermentation product that is unknown whether it contains lactic acid bacteria and a fermentation product that is known to contain lactic acid bacteria; The fermented products, whether or not they contain lactic acid bacteria, include kimchi, yogurt, and silage; When the sample is a fermentation product of unknown lactic acid bacteria content, the activation and enrichment process involves adding the fermentation product of unknown lactic acid bacteria content to a modified liquid culture medium, culturing it under anaerobic conditions at 37°C for 18 hours, streaking it onto a modified solid culture medium with an inoculation loop, culturing it under anaerobic conditions at 37°C for 24 hours, then picking a single colony and adding it to 5 mL of modified liquid culture medium, culturing it under anaerobic conditions at 37°C for 18 hours to obtain the activated and enriched bacterial suspension. When the sample is a fermentation product known to contain lactic acid bacteria, the activation and enrichment process involves inoculating the fermentation product containing lactic acid bacteria at an inoculation amount of 2% into a modified liquid culture medium, and incubating it at 37°C for 18 hours to obtain an activated and enriched bacterial suspension. The modified liquid culture medium consists of: peptone 9-11 g / L, yeast extract 4.5-5.5 g / L, lactose 18-20 g / L, triammonium citrate 0.5-0.6 g / L, manganese sulfate 0.24-0.26 g / L, anhydrous sodium acetate 1.8-1.9 g / L, disodium hydrogen phosphate 1.6-1.7 g / L, potassium acetate 2.2-2.3 g / L, beef extract 9-11 g / L, bovine bile salts 0.9-1.1 g / L, L-cysteine ​​0.4-0.6 g / L, and Tween-80 0.9-1.1 mL / L.

[0007] The modified solid culture medium consists of: peptone 9-11 g / L, yeast extract 4.5-5.5 g / L, lactose 18-20 g / L, triammonium citrate 0.5-0.6 g / L, manganese sulfate 0.24-0.26 g / L, anhydrous sodium acetate 1.8-1.9 g / L, disodium hydrogen phosphate 1.6-1.7 g / L, potassium acetate 2.2-2.3 g / L, beef extract 9-11 g / L, ox bile salts 0.9-1.1 g / L, L-cysteine ​​0.4-0.6 g / L, Tween-80 0.9-1.1 mL / L, and agar powder 14-16 g / L.

[0008] The preparation of the single-cell suspension involves centrifuging the activated and enriched bacterial suspension, collecting the bacterial cells, washing, adjusting the cell concentration, and obtaining the single-cell suspension. In the preparation of the single-cell suspension, the centrifugation speed is 6000-8000 r / min and the centrifugation time is 3-5 min; Use sterile PBS buffer for washing, and wash 3-5 times. When adjusting cell concentration, resuspend the cells using sterile PBS buffer. OD of the single-cell suspension 600 The value is 0.9-1.1; The assessment of the physiological state and activity of the cells includes: nucleic acid double staining, cell membrane integrity assessment, cell metabolic activity assessment, and cell membrane lipid peroxidation assessment. The nucleic acid double staining includes: staining with SYBR Green II and staining with PI; The cell membrane integrity was assessed using DiBAC4(3) staining; The cell metabolic activity was assessed using FDA staining. The assessment of cell membrane lipid peroxidation was performed using BODIPY 581 / 591 C11 staining; The flow cytometry analysis includes: parameter setting, data acquisition and analysis, and comprehensive activity scoring; The parameter settings use 488nm and 561nm lasers as excitation sources. The green fluorescence in the fluorescence detection channel is used to monitor the SYBR Green II and FDA signals, and is detected using a 530 / 30nm bandpass filter. The red fluorescence is used to monitor the PI signal, and is detected using a 610 / 20nm bandpass filter. The orange fluorescence is used to monitor the DiBAC4(3) signal, and is detected using a 585 / 42nm bandpass filter. The red fluorescence is used to monitor the BODIPY 581 / 591 C11 signal, and is detected using a 630 / 20nm bandpass filter. The data acquisition and analysis include: total cell gating, live cell gating, cell membrane integrity assessment, cell metabolic activity assessment, and cell membrane lipid peroxidation assessment. The total cell gating is distinguished by SYBR Green II signal; the live cell gating is distinguished by SYBR Green II signal and PI signal; the cell membrane integrity assessment is distinguished by DiBAC4(3) signal; the cell metabolic activity assessment is distinguished by FDA signal; the cell membrane lipid peroxidation assessment is distinguished by BODIPY 581 / 591 C11 signal. The comprehensive activity score is determined by data collection and analysis results to screen for highly active cells. The comprehensive activity score is based on live cell gating, and the comprehensive activity score = (live cell gating score × 0.4) + (membrane integrity assessment score × 0.25) + (metabolic activity assessment score × 0.2) + (cell membrane lipid peroxidation assessment score × 0.15). Cells with a comprehensive activity score ≥80 points, and individual index scores of membrane integrity assessment ≥60 points, metabolic activity assessment ≥70 points, and cell membrane lipid peroxidation assessment ≥50 points are defined as highly active cells. When the sample with concentrated bacterial cells is a fermentation product known to contain lactic acid bacteria, the cell sorting and bacterial species identification involves PCR amplification of highly active cells, 16S rRNA sequencing, and determination of the bacterial species. When the sample with concentrated bacterial cells is a fermentation product of unknown origin, the cell sorting and bacterial species identification are performed by PCR amplification of highly active lactic acid bacteria cells, followed by 16S rRNA sequencing to determine the strain species. The screening method for highly active lactic acid bacteria cells is to perform lactic acid bacteria-specific screening on highly active cells; The specific screening of lactic acid bacteria includes: lactic acid detection, extracellular polysaccharide detection, and comprehensive activity score; The lactic acid detection was performed by staining with FITC-lactic acid dye, and FITC-lactic acid evaluation was conducted at an excitation wavelength of 490 nm and an emission wavelength of 520 nm to obtain a FITC-lactic acid evaluation score. The extracellular polysaccharide detection was performed by staining with FITC-ConA dye, and FITC-ConA evaluation was conducted at an excitation wavelength of 490 nm and an emission wavelength of 520 nm to obtain the FITC-ConA evaluation score. The comprehensive activity score is based on the FITC-lactic acid assessment score and the FITC-ConA assessment score to screen for highly active lactic acid bacteria cells. In the comprehensive activity score, the comprehensive activity score = (FITC-lactic acid assessment score × 0.7) + (FITC-ConA assessment score × 0.3). Cells with a comprehensive score of ≥80 points, and individual indicator scores of ≥75 points for FITC-lactic acid assessment and ≥60 points for FITC-ConA assessment, are defined as highly active lactic acid bacteria cells.

[0009] An application of the aforementioned method in screening highly viable lactic acid bacteria.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method of high-throughput screening of high-activity lactic acid bacteria based on multi-index fusion of the present invention provides a method for detecting the activity of fermentation products and long-term stored lactic acid bacteria. The method of the present invention is convenient and fast, greatly shortens the screening time and improves the screening efficiency.

[0011] (2) The high-throughput screening method for high-activity lactic acid bacteria based on multi-index fusion of the present invention combines multiple methods for comprehensive evaluation, which can effectively solve the limitations of single evaluation; the authenticity of cell structure survival is verified by nucleic acid double staining and cell membrane integrity assessment; the energy metabolism status of living cells is judged by cell metabolic activity assessment; the stress tolerance of cells is analyzed by lipid peroxidation assessment; and finally, the direct correlation between functional products and living cell metabolism is indicated by lactic acid and extracellular polysaccharide detection. This multi-method combined evaluation not only eliminates false positive / false negative interference, but also truly constructs a "high activity" evaluation standard that fits the actual application of lactic acid bacteria.

[0012] (3) The high-throughput screening method for high-activity lactic acid bacteria based on multi-index fusion of the present invention uses modified liquid culture medium and modified solid culture medium to enrich the strains before screening, which can promote bacterial growth and shorten bacterial growth time.

[0013] (4) The high-throughput screening method for high-activity lactic acid bacteria based on multi-index fusion of the present invention constructs a cell activity assessment model through the above multi-index fusion analysis. The activity scoring assessment model is to score the activity of each cell according to the weight allocation of different indicators. The accuracy (95-98%) of the combination of 4 indicators, namely, live cell gating, cell membrane integrity assessment, cell metabolic activity assessment, and cell membrane lipid peroxidation assessment, is significantly higher than that of a single indicator. Moreover, the stability of the combination of 4 indicators is above 95%, which shortens the screening time by 72.2%. Attached Figure Description

[0014] Figure 1 This is a flowchart of Example 1. Detailed Implementation

[0015] The technical solution of the present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto. Contents not described in detail in the embodiments are based on existing technology in the field.

[0016] Example 1 The culture medium and its components used in this embodiment are as follows: The modified liquid culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 20 g / L lactose, 0.58 g / L triammonium citrate, 0.25 g / L manganese sulfate, 1.88 g / L anhydrous sodium acetate, 1.63 g / L disodium hydrogen phosphate, 2.25 g / L potassium acetate, 10 g / L beef extract, 1 g / L ox bile salts, 0.5 g / L L-cysteine, and 1 mL / L Tween-80.

[0017] The modified solid culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 20 g / L lactose, 0.58 g / L triammonium citrate, 0.25 g / L manganese sulfate, 1.88 g / L anhydrous sodium acetate, 1.63 g / L disodium hydrogen phosphate, 2.25 g / L potassium acetate, 10 g / L beef extract, 1 g / L ox bile salts, 0.5 g / L L-cysteine, 1 mL / L Tween-80, and 15 g / L agar powder.

[0018] The composition of the MRS liquid culture medium is as follows: peptone 10 g / L, yeast extract 5 g / L, glucose 20 g / L, triammonium citrate 0.58 g / L, manganese sulfate 0.25 g / L, anhydrous sodium acetate 1.88 g / L, disodium hydrogen phosphate 1.63 g / L, potassium acetate 2.25 g / L, beef extract 10 g / L, and Tween-80 1 mL / L.

[0019] The MRS solid culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 20 g / L glucose, 0.58 g / L triammonium citrate, 0.25 g / L manganese sulfate, 1.88 g / L anhydrous sodium acetate, 1.63 g / L disodium hydrogen phosphate, 2.25 g / L potassium acetate, 10 g / L beef extract, 1 mL / L Tween-80, and 15 g / L agar powder.

[0020] This embodiment presents a method for high-throughput screening of highly active lactic acid bacteria based on multi-index fusion, according to... Figure 1 The flowchart is as follows: 1. Bacterial cell enrichment Samples were collected from naturally fermented kimchi and added to modified liquid culture medium. The samples were cultured anaerobically at 37°C for 18 hours. The samples were then streaked onto modified solid culture medium using an inoculation loop and cultured anaerobically at 37°C for 24 hours. Single colonies were then picked and added to 5 mL of modified liquid culture medium and cultured anaerobically at 37°C for 18 hours for activation and enrichment, resulting in an activated and enriched bacterial suspension. Meanwhile, to verify the promoting effect of modified liquid culture medium and modified solid culture medium on the growth of lactic acid bacteria, a comparative experiment was set up, specifically as follows: MRS culture medium group: Samples were collected from naturally fermented kimchi and cultured in MRS liquid medium at 37°C for 18 hours under anaerobic conditions. The samples were then streaked onto MRS solid medium and cultured at 37°C for 24 hours under anaerobic conditions. Single colonies were then picked and cultured in 5 ml MRS liquid medium at 37°C under anaerobic conditions. OD values ​​were measured at 3, 6, 9, 12, 15, 18, 21, and 24 hours after culture. 600 And pH were measured; Modified culture medium group: Samples were collected from naturally fermented kimchi and placed in modified liquid culture medium. The samples were cultured anaerobically at 37°C for 18 hours. Then, samples were streaked onto modified solid culture medium and cultured anaerobically at 37°C for 24 hours. Single colonies were then picked and placed into 5 mL of modified liquid culture medium and cultured anaerobically at 37°C. OD values ​​were measured at 3, 6, 9, 12, 15, 18, 21, and 24 hours after culture. 600 And pH were measured; The specific test results are shown in Table 1: Table 1

[0021] Table 1 shows that, compared with the MRS medium culture group, the OD of the modified medium culture group was higher. 600 The rapid pH changes indicate that the modified liquid and solid culture media can shorten the bacterial growth time.

[0022] 2. Preparation of single-cell suspension The activated and enriched bacterial suspension was centrifuged (6000 r / min, 3 min) to collect the bacterial cells. The cells were washed three times with sterile PBS buffer and then adjusted to the OD value with sterile PBS buffer. 600 =1, and a single-cell suspension was prepared.

[0023] 3. Assessment of cellular physiological state and activity (1) Double staining of nucleic acids ①SYBR Green II staining: Take 1 mL of single cell suspension, add 1 μL of SYBR Green II dye working solution (final concentration 1×), mix gently, and incubate at room temperature in the dark for 5 min to obtain SYBR Green II stained cell suspension.

[0024] ②PI staining: Add 1 μL of PI dye working solution (final concentration 1×) to the SYBR Green II stained cell suspension, mix gently, and incubate at room temperature in the dark for 5 min to obtain a nucleic acid double-stained cell suspension.

[0025] (2) Assessment of cell membrane integrity DiBAC4(3) staining: Add 5 μL of DiBAC4(3) dye working solution (final concentration 5 μM) to the cell suspension with nucleic acid double staining, mix gently, and incubate at room temperature in the dark for 10 minutes to obtain DiBAC4(3) stained cell suspension.

[0026] (3) Assessment of cellular metabolic activity FDA staining: Add 2 μL of FDA dye working solution (final concentration 10 μg / mL) to the DiBAC4(3) stained cell suspension, mix gently, and incubate at 37°C in the dark for 15 minutes to obtain FDA stained cell suspension.

[0027] (4) Assessment of cell membrane lipid peroxidation BODIPY 581 / 591 C11 staining: Add 1 μL of BODIPY 581 / 591C11 dye working solution (final concentration 1 μM) to the FDA-stained cell suspension, mix gently, and incubate at 37°C in the dark for 30 minutes to obtain the stained cell suspension.

[0028] 4. Flow cytometry analysis The stained cell suspension was analyzed by flow cytometry. The analytical method is as follows: (1) Set the instrument parameters as follows: Excitation source: 488nm and 561nm lasers were used as excitation sources.

[0029] Fluorescence detection channels: Green fluorescence (SYBR Green II and FDA): detected using a 530 / 30nm bandpass filter; Red fluorescence (PI): detected using a 610 / 20nm bandpass filter; Orange fluorescence (DiBAC4(3)): detected using a 585 / 42nm bandpass filter; Red fluorescence (BODIPY 581 / 591 C11): detected using a 630 / 20nm bandpass filter.

[0030] (2) Data collection and analysis: ① Total cell gating: Gating was performed using the SYBR Green II signal to distinguish all bacterial cells.

[0031] ② Live cell gating: Combining SYBR Green II and PI signals, it distinguishes between live cells (which emit only green fluorescence) and dead cells (which emit red fluorescence or both green and red fluorescence).

[0032] ③ Cell membrane integrity assessment: The DiBAC4(3) signal was used to assess the changes in membrane potential of live cells, and to further distinguish between cells with intact membranes and cells with damaged membranes.

[0033] ④ Cellular metabolic activity assessment: Using FDA signals, assess the metabolic activity of live cells and distinguish between cells with high metabolic activity and cells with low metabolic activity.

[0034] ⑤ Assessment of cell membrane lipid peroxidation: The degree of lipid peroxidation in live cell membranes was assessed using the BODIPY 581 / 591 C11 signal, and cells with different degrees of membrane lipid peroxidation were distinguished.

[0035] The cells were comprehensively scored, and the scoring criteria are shown in Table 2: Table 2

[0036] Live cell gating is the basis for all subsequent scoring. The overall activity score = (live cell gating score × 0.4) + (membrane integrity assessment score × 0.25) + (metabolic activity assessment score × 0.2) + (cell membrane lipid peroxidation assessment score × 0.15) Cells with a comprehensive activity score of ≥80 points, and whose individual index scores include ≥60 points for membrane integrity assessment, ≥70 points for metabolic activity assessment, and ≥50 points for cell membrane lipid peroxidation assessment, are defined as highly active cells.

[0037] Highly active cells were selected based on the comprehensive activity score.

[0038] The screening efficiency of different fluorescence indicators is shown in Table 3: Table 3

[0039] The formulas for calculating the live cell detection rate, false positive rate, and stability in the table above are as follows: Live cell detection rate (the proportion of actual live cells correctly identified as live cells) = [Fluorescently labeled as live cells, actually live cells / (Fluorescently labeled as live cells, actually live cells + Fluorescently labeled as dead cells, actually live cells)] × 100%; False positive rate (the proportion of dead cells incorrectly identified as live cells) = [Fluorescently labeled live cells that are actually dead cells / (Fluorescently labeled live cells that are actually dead cells + Fluorescently labeled dead cells that are actually dead cells)] × 100%; Stability (the ability of fluorescence indicators to maintain consistent results over a long period of time) = 1 - [(initial viable cell detection rate - endpoint viable cell detection rate) / initial viable cell detection rate] × 100%.

[0040] 5. Specific screening of lactic acid bacteria (1) Lactate detection: Dilute FITC-lactic acid dye to 10 μg / mL with PBS, then resuspend the highly active cells from Example 3 in PBS and mix with an equal volume of FITC-lactic acid staining solution to obtain a mixture, ensuring that the cell concentration in the mixture is 1×10⁻⁶. 6 The mixture was incubated at CFU / mL at 37°C in the dark for 30 min to ensure that the dye fully interacted with the cells. After incubation, the cells were washed three times with PBS to remove unbound FITC-lactic acid dye.

[0041] Then, adjust the laser wavelength and filter, setting the excitation wavelength to 490nm and the emission wavelength to 520nm to match the fluorescence characteristics of FITC, and then sort them on the machine.

[0042] (2) Detection of extracellular polysaccharides (EPS): FITC-ConA dye was diluted to 10 μg / mL with PBS. The highly active cells from Example 3 were resuspended in PBS and mixed with an equal volume of FITC-lactic acid staining solution to obtain a mixture, ensuring that the cell concentration in the mixture was 1×10⁻⁶. 6 The mixture was incubated at CFU / mL at 37°C in the dark for 30 minutes to ensure that the dye fully interacted with the cells. After incubation, the cells were washed three times with PBS to remove unbound FITC-ConA dye.

[0043] Adjust the laser wavelength and filter, set the excitation wavelength to 490nm and the emission wavelength to 520nm to match the fluorescence characteristics of FITC, and then sort them on the machine.

[0044] The above-mentioned FITC-lactic acid and FITC-ConA were used for specific screening of lactic acid bacteria, and the scoring criteria are shown in Table 4. Table 4

[0045] Overall activity score = (FITC-lactic acid assessment score × 0.7) + (FITC-ConA assessment score × 0.3) Cells with a comprehensive score of ≥80 points, and individual indicator scores of ≥75 points for FITC-lactic acid assessment and ≥60 points for FITC-ConA assessment, are defined as highly active lactic acid bacteria cells.

[0046] By using the combined use of FITC-lactic acid and FITC-ConA for lactic acid bacteria specific screening, the positive rate after screening results verification was higher than that of the traditional plate method and the use of FITC-lactic acid and FITC-ConA alone. At the same time, the detection time was significantly shortened compared to the traditional plate method. The comparison results with the traditional plate method and the use of FITC-lactic acid and FITC-ConA alone are shown in Table 5. Table 5

[0047] The formulas for calculating the positive rate and true positive rate in the table above are as follows: Positive rate = (Number of positive strains / Total number of strains) × 100%; True positive rate = (Number of target strains after validation / Total number of positive strains) × 100%.

[0048] 6. Cell sorting and strain identification Single colonies were taken from the highly active lactic acid bacteria cells obtained in step 5 and inoculated into modified solid medium. The culture was incubated at 37°C for 18 hours. Then, a 1% (v / v) inoculum was inoculated into fresh, sterile modified liquid medium and incubated at 37°C for another 18 hours. One mL of the bacterial culture was taken from the liquid medium, and the genome was extracted according to the instructions of the Nanjing Novizan Biotechnology Co., Ltd. genome extraction kit. Using the extracted genome as a template, PCR amplification was performed using primers 27F and 1492R. The primer sequences and amplification program for PCR amplification are shown in Tables 6 and 7. Table 6

[0049] Table 7

[0050] The amplification products were verified by agarose gel electrophoresis. PCR products with correct agarose gel electrophoresis bands were selected for 16S rRNA sequencing. The sequencing results were compared with those from the National Center for Biotechnology Information (NCBI) to determine the strain species.

[0051] Example 2 The culture medium and its components used in this embodiment are as follows: The modified liquid culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 20 g / L lactose, 0.58 g / L triammonium citrate, 0.25 g / L manganese sulfate, 1.88 g / L anhydrous sodium acetate, 1.63 g / L disodium hydrogen phosphate, 2.25 g / L potassium acetate, 10 g / L beef extract, 1 g / L ox bile salts, 0.5 g / L L-cysteine, and 1 mL / L Tween-80.

[0052] The modified solid culture medium consists of: 10 g / L peptone, 5 g / L yeast extract, 20 g / L lactose, 0.58 g / L triammonium citrate, 0.25 g / L manganese sulfate, 1.88 g / L anhydrous sodium acetate, 1.63 g / L disodium hydrogen phosphate, 2.25 g / L potassium acetate, 10 g / L beef extract, 1 g / L ox bile salts, 0.5 g / L L-cysteine, 1 mL / L Tween-80, and 15 g / L agar powder.

[0053] The composition of the MRS liquid culture medium is as follows: peptone 10 g / L, yeast extract 5 g / L, glucose 20 g / L, triammonium citrate 0.58 g / L, manganese sulfate 0.25 g / L, anhydrous sodium acetate 1.88 g / L, disodium hydrogen phosphate 1.63 g / L, potassium acetate 2.25 g / L, beef extract 10 g / L, and Tween-80 1 mL / L.

[0054] Similarly, lactic acid bacteria stored for a long time will have reduced activity due to prolonged storage. Using the method in Example 1 for rapid screening can greatly shorten the screening time and improve screening efficiency.

[0055] This embodiment provides a high-throughput screening method for highly viable lactic acid bacteria based on multi-index fusion. The screening target is lactic acid bacteria that have been stored for a long time. Figure 1 The flowchart is as follows: Long-term stored lactic acid bacteria culture was inoculated at an inoculum size of 2% into fresh, sterile modified liquid culture medium and incubated statically at 37°C for 18 hours to obtain a bacterial suspension. The bacterial suspension was centrifuged (6000 r / min, 3 min) to collect the bacterial cells, washed three times with sterile PBS buffer, and the cell concentration was adjusted to OD using sterile PBS buffer. 600 =1, prepare a single-cell suspension, stain and evaluate the bacterial suspension according to the staining method in step 3 of Example 1, which is the assessment of the physiological state and activity of cells, and then analyze it according to step 4, which is the flow cytometry analysis, and screen to obtain highly viable cells; Single colonies of highly viable cells obtained from screening were inoculated into modified solid medium and cultured at 37°C for 18 h. Then, they were inoculated into fresh, sterile modified liquid medium at a volume fraction of 1% and cultured at 37°C for 18 h. 1 mL of the bacterial culture was taken from the liquid medium and the genome was extracted according to the instructions of the genome extraction kit from Nanjing Novizan Biotechnology Co., Ltd. The extracted genome was used as a template for PCR amplification with primers 27F and 1492R to ensure the correctness of the bacterial strain.

[0056] Simultaneously, take 1 mL of bacterial culture, centrifuge at 6000 r / min for 3 min, discard the supernatant, resuspend the bacterial cells in 1 mL of fresh sterile MRS liquid medium, and mix with 50% glycerol at a volume ratio of 1:1 to obtain a new bacterial culture glycerol tube that can be stored for a long time.

[0057] The method in this embodiment can screen hundreds of strains at once, shortening the screening time and improving screening efficiency.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for high-throughput screening of highly viable lactic acid bacteria based on multi-index fusion, characterized in that, include: Bacterial cell enrichment, preparation of single-cell suspensions, assessment of cell physiological state and activity, flow cytometry analysis, cell sorting and bacterial species identification; The bacterial cell enrichment was achieved by activating and enriching the sample using modified liquid culture medium and modified solid culture medium to obtain an activated and enriched bacterial suspension. The modified liquid culture medium consists of: peptone 9-11 g / L, yeast extract 4.5-5.5 g / L, lactose 18-20 g / L, triammonium citrate 0.5-0.6 g / L, manganese sulfate 0.24-0.26 g / L, anhydrous sodium acetate 1.8-1.9 g / L, disodium hydrogen phosphate 1.6-1.7 g / L, potassium acetate 2.2-2.3 g / L, beef extract 9-11 g / L, bovine bile salts 0.9-1.1 g / L, L-cysteine ​​0.4-0.6 g / L, and Tween-80 0.9-1.1 mL / L. The modified solid culture medium consists of: peptone 9-11 g / L, yeast extract 4.5-5.5 g / L, lactose 18-20 g / L, triammonium citrate 0.5-0.6 g / L, manganese sulfate 0.24-0.26 g / L, anhydrous sodium acetate 1.8-1.9 g / L, disodium hydrogen phosphate 1.6-1.7 g / L, potassium acetate 2.2-2.3 g / L, beef extract 9-11 g / L, ox bile salts 0.9-1.1 g / L, L-cysteine ​​0.4-0.6 g / L, Tween-80 0.9-1.1 mL / L, and agar powder 14-16 g / L.

2. The method for high-throughput screening of highly viable lactic acid bacteria based on multi-index fusion according to claim 1, characterized in that, The bacterial cells are enriched, and the sample is a fermentation product that is unknown whether it contains lactic acid bacteria and a fermentation product that is known to contain lactic acid bacteria; The fermented products, whether or not they contain lactic acid bacteria, include kimchi, yogurt, and silage; When the sample is a fermentation product of unknown lactic acid bacteria content, the activation and enrichment process involves adding the fermentation product of unknown lactic acid bacteria content to a modified liquid culture medium, culturing it under anaerobic conditions at 37°C for 18 hours, streaking it onto a modified solid culture medium with an inoculation loop, culturing it under anaerobic conditions at 37°C for 24 hours, then picking a single colony and adding it to 5 mL of modified liquid culture medium, culturing it under anaerobic conditions at 37°C for 18 hours to obtain the activated and enriched bacterial suspension. When the sample is a fermentation product known to contain lactic acid bacteria, the activation and enrichment process involves inoculating the fermentation product containing lactic acid bacteria at an inoculum amount of 2% into a modified liquid culture medium and incubating it at 37°C for 18 hours to obtain an activated and enriched bacterial suspension.

3. The method for high-throughput screening of highly viable lactic acid bacteria based on multi-index fusion according to claim 1, characterized in that, The preparation of the single-cell suspension involves centrifuging the activated and enriched bacterial suspension, collecting the bacterial cells, washing, adjusting the cell concentration, and obtaining the single-cell suspension. In the preparation of the single-cell suspension, the centrifugation speed is 6000-8000 r / min and the centrifugation time is 3-5 min; Use sterile PBS buffer for washing, and wash 3-5 times. When adjusting cell concentration, resuspend the cells using sterile PBS buffer. OD of the single-cell suspension 600 It ranges from 0.9 to 1.

1.

4. The method for high-throughput screening of highly viable lactic acid bacteria based on multi-index fusion according to claim 1, characterized in that, The assessment of the physiological state and activity of the cells includes: nucleic acid double staining, cell membrane integrity assessment, cell metabolic activity assessment, and cell membrane lipid peroxidation assessment. The nucleic acid double staining includes: staining with SYBR Green II and staining with PI; The cell membrane integrity was assessed using DiBAC4(3) staining; The cell metabolic activity was assessed using FDA staining. The assessment of cell membrane lipid peroxidation was performed using BODIPY 581 / 591 C11 staining.

5. The method for high-throughput screening of highly viable lactic acid bacteria based on multi-index fusion according to claim 1, characterized in that, The flow cytometry analysis includes: parameter setting, data acquisition and analysis, and comprehensive activity scoring; The parameter settings use 488nm and 561nm lasers as excitation sources. The green fluorescence in the fluorescence detection channel is used to monitor the SYBR Green II and FDA signals, and is detected using a 530 / 30nm bandpass filter. The red fluorescence is used to monitor the PI signal, and is detected using a 610 / 20nm bandpass filter. The orange fluorescence is used to monitor the DiBAC4(3) signal, and is detected using a 585 / 42nm bandpass filter. The red fluorescence is used to monitor the BODIPY 581 / 591 C11 signal, and is detected using a 630 / 20nm bandpass filter. The data acquisition and analysis include: total cell gating, live cell gating, cell membrane integrity assessment, cell metabolic activity assessment, and cell membrane lipid peroxidation assessment. The total cell gating is distinguished by SYBR Green II signal; the live cell gating is distinguished by SYBR Green II signal and PI signal; the cell membrane integrity assessment is distinguished by DiBAC4(3) signal; the cell metabolic activity assessment is distinguished by FDA signal; the cell membrane lipid peroxidation assessment is distinguished by BODIPY 581 / 591C11 signal. The comprehensive activity score is determined by data collection and analysis results to screen for highly active cells.

6. The method for high-throughput screening of highly viable lactic acid bacteria based on multi-index fusion according to claim 1, characterized in that, When the sample with concentrated bacterial cells is a fermentation product known to contain lactic acid bacteria, the cell sorting and bacterial species identification involves PCR amplification of highly active cells, 16S rRNA sequencing, and determination of the bacterial species. When the sample with concentrated bacterial cells is a fermentation product of unknown origin, the cell sorting and bacterial species identification involve PCR amplification of highly active lactic acid bacteria cells, 16S rRNA sequencing, and determination of the strain species.

7. The method for high-throughput screening of highly viable lactic acid bacteria based on multi-index fusion according to claim 6, characterized in that, The screening method for highly active lactic acid bacteria cells is to perform lactic acid bacteria-specific screening on highly active cells; The specific screening of lactic acid bacteria includes: lactic acid detection, extracellular polysaccharide detection, and comprehensive activity score; The lactic acid detection was performed by staining with FITC-lactic acid dye, and FITC-lactic acid evaluation was conducted at an excitation wavelength of 490 nm and an emission wavelength of 520 nm to obtain a FITC-lactic acid evaluation score. The extracellular polysaccharide detection was performed by staining with FITC-ConA dye, and FITC-ConA evaluation was conducted at an excitation wavelength of 490 nm and an emission wavelength of 520 nm to obtain the FITC-ConA evaluation score. The comprehensive activity score is based on the FITC-lactic acid assessment score and the FITC-ConA assessment score to screen for highly active lactic acid bacteria cells.

8. The application of the method according to any one of claims 1-7 in screening for highly viable lactic acid bacteria.

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