Method for counting dead cells of lactic acid bacteria in metabiotics

Abstract

The invention belongs to the technical field of cell counting, and particularly relates to a method for counting dead cells of lactic acid bacteria in postbiotics. The method for counting dead lactic acid bacteria cells in the metagen comprises the following steps: sample pretreatment: taking to-be-detected metagen bacterial powder, and preparing a sample bacterial suspension; counting by flow cytometry: diluting the sample bacterium suspension to 1.0 * 10 < 6 > CFU / mL to 1.0 * 10 < 7 > CFU / mL, adding two fluorescent dyes, and incubating at room temperature in a dark environment for 20-30 minutes to obtain a double-dyed sample; taking the double-dyed sample, and carrying out quantity analysis and detection by adopting a flow cytometry to obtain the quantity of the dead lactic acid bacteria cells in the postbiotics; the fluorescent dye is prepared from DRAQ7 and calcein AM; the final concentration of the DRAQ7 fluorescent dye ranges from 2.8 M to 3.2 M, and the final concentration of the calcein AM ranges from 4.8 M to 5.2 M. The invention provides a method for counting dead cells of lactic acid bacteria in a metagen. The method is a convenient and rapid quantitative method for cells in the metagen.

Description

Technical Field

[0001] The invention belongs to the technical field of cell counting, and particularly relates to a method for counting dead lactic acid bacteria cells in postbiotics. Background Art

[0002] The International Scientific Association of Probiotics and Prebiotics (ISAPP) defines postbiotics as "preparations of non-living microorganisms and / or their components that have a health benefit to the host." This definition states that effective postbiotics, regardless of whether they contain bacterial metabolites, should be ingredients with well-defined health effects, including strains of bacteria recognized as safe and with a well-defined genetic background and biological characteristics, inactivated microorganisms, bacterial components, and metabolites. This excludes ingredients such as metabolites of purified viral (including bacteriophage) strains, vaccines, and traditional fermentation products with undefined bacterial composition.

[0003] Postbiotics not only retain the original properties of probiotics, but also lack their ability to grow and reproduce. They are unaffected by temperature and environmental influences, making them easier to store and transport, and their properties more stable. As a mixture of several components of inactivated probiotics and bacterial components, quality control and monitoring of postbiotics can only be achieved by identifying their characteristic components that impact health and establishing qualitative and quantitative analytical methods.

[0004] Currently, methods for counting cells in postbiotics use commonly used methods for counting microbial cells. Whether using hemocytometers, plate pour counts, direct microscopic counting, turbidimetry, or membrane filtration methods, most methods count live cells or total bacteria to infer the number of dead lactic acid bacteria cells in postbiotics. These methods are unable to accurately count dead lactic acid bacteria cells in postbiotics. Therefore, the commonly used microbial cell counting methods currently used are not suitable for counting dead lactic acid bacteria cells in postbiotics. Summary of the Invention

[0005] To address the problem that conventional microbial cell counting methods currently used in the prior art are unsuitable for counting dead lactic acid bacteria cells in postbiotics, the present invention uses flow cytometry to count dead cells of probiotic postbiotic lactic acid bacteria, and compares the results with plate pour counts to develop a convenient and rapid method for counting dead lactic acid bacteria cells in postbiotics. To achieve this objective, the present invention employs the following technical solutions.

[0006] A method for counting dead lactic acid bacteria cells in postbiotics comprises the following steps: Sample pretreatment: Take the postbiotic bacterial powder to be tested and prepare the sample bacterial suspension.

[0007] Flow cytometry counting: dilute the sample bacterial suspension to 1.0×10 6 CFU / mL~1.0×107 CFU / mL, add at least two fluorescent dyes, and incubate in the dark at room temperature to obtain a double-stained sample. The double-stained sample is taken and quantitatively analyzed and detected using flow cytometry to determine the number of dead lactic acid bacteria cells in the postbiotic. The fluorescent dyes include DRAQ7 fluorescent dye and calcein AM. The final concentration of the DRAQ7 fluorescent dye is 2.8µM to 3.2µM, and the final concentration of the calcein AM is 4.8µM to 5.0µM.

[0008] The present invention provides a method for counting dead lactic acid bacteria cells in postbiotics, which includes sample pretreatment and flow cytometry counting; sample pretreatment includes the preparation of a sample bacterial suspension; the core advantage of flow cytometry counting is its accuracy, efficiency, and automation, which perfectly meets the quantitative requirements of dead lactic acid bacteria in postbiotics. The method provided by the present invention is a method suitable for counting dead lactic acid bacteria cells in postbiotics, and is a convenient and rapid method for quantifying cells in postbiotics. It can solve the problem that the commonly used counting methods for microbial cells currently used in the prior art are not suitable for counting dead lactic acid bacteria cells in postbiotics.

[0009] More preferably, the final concentration of the DRAQ7 fluorescent dye is 3.0 μM, and the final concentration of the calcein is 5.0 μM.

[0010] Preferably, the incubation time at room temperature in the dark is 20 to 30 minutes. By controlling the incubation time, temperature, and environmental conditions, the target reaction can be promoted more fully and efficiently, thereby improving the accuracy and reliability of the experimental results.

[0011] Preferably, the preparation method of the sample bacterial suspension is as follows: Take the postbiotic powder to be tested and dilute it with a diluent under a sterile environment to obtain the sample bacterial suspension. Dilute it to an appropriate bacterial concentration so that subsequent experiments can be carried out accurately and efficiently, avoiding excessively high or low bacterial concentrations.

[0012] Preferably, the diluent is a PBS buffer solution; the PBS buffer solution is composed of the following effective substances at the following final concentrations: Sodium chloride 7.8g / L~8.2g / L, disodium hydrogen phosphate 1.13g / L~1.17g / L and potassium dihydrogen phosphate 0.1g / L~0.3g / L, the solvent is water.

[0013] Preferably, the postbiotic bacterial powder to be tested is obtained by inactivating the strain to be tested and then subjecting it to vacuum freeze-drying.

[0014] Preferably, the strain to be tested includes at least one of Lactobacillus paracasei PC-01, Bifidobacterium animalis subsp. lactis BX-246, Lactobacillus paracasei PC-92, and Lactobacillus rhamnosus Prosci-109. These bacteria are common core probiotics and are Gram-positive bacteria. The cell wall structure of Gram-positive bacteria significantly affects dye permeability, and after staining, the fluorescence signals of live and dead cells are clearly differentiated, facilitating flow cytometric analysis.

[0015] Preferably, the inactivation condition is 94°C to 96°C for 10 min to 20 min.

[0016] The vacuum freeze-drying conditions are as follows: a minimum temperature of -35°C to 45°C, a vacuum degree of 14 MPa to 15 MPa, and a pre-freezing time of 2.8 hours to 3.2 hours.

[0017] Preferably, before quantitative analysis and detection using flow cytometry, an equal volume of absolute counting microspheres is added to the double-stained sample and then mixed.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for counting dead cells of lactic acid bacteria in postbiotics, which is a convenient and rapid method for quantifying cells in postbiotics. The method includes sample pretreatment and flow cytometry counting; sample pretreatment includes the preparation of a sample bacterial suspension; the core advantage of flow cytometry counting is that it is accurate, efficient, and automated, and perfectly adapted to the quantitative requirements of dead cells in postbiotics. The method provided by the present invention is a method suitable for counting dead cells of lactic acid bacteria in postbiotics, and is a convenient and rapid method for quantifying cells in postbiotics, which can solve the problem that the commonly used counting methods for microbial cells currently used in the prior art are not suitable for counting dead cells of lactic acid bacteria in postbiotics.

[0019] 2. The present invention uses postbiotics with probiotic properties screened in the early laboratory as the research object, optimizes the fluorescent dye staining time and concentration combination through orthogonal experiments, determines its staining time and optimal concentration combination, and uses flow cytometry to count dead lactic acid bacteria cells in postbiotics. The results are compared with traditional plate pour counting to develop a more convenient and rapid method for counting dead lactic acid bacteria cells in postbiotics.

[0020] 3. The present invention first dilutes the postbiotic powder to 10 6 CFU / mL~10 7CFU / mL, stained with DRAQ7 and calcein fluorescent dyes, and the number of dead cells was detected using a flow cytometer; at the same time, traditional plate colonies were used to count the bacteria before inactivation. The verification results determined that the optimal staining conditions were a staining time of 25 minutes, a dye concentration combination of a final concentration of DRAQ7 fluorescent dye of 3.0µM, and a final concentration of calcein of 5.0µM. Under these conditions, there was no significant difference in the postbiotic counting results between flow cytometry and the plate pouring counting method, indicating that this method is suitable for counting dead lactic acid bacteria cells in postbiotics. The present invention provides a method for quantitative analysis of dead lactic acid bacteria cells in postbiotics, in order to lay the foundation for providing theoretical guidance for the quality control and supervision of postbiotics. DETAILED DESCRIPTION

[0021] The following specific examples are provided to illustrate the present invention in detail, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0022] The experimental materials used in the following examples are as follows: 1. Source of strain The postbiotic powder used in this experiment was prepared by the Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University. Lacticaseibacillus paracasei PC-01), Bifidobacterium animalis subsp. lactis BX-246 ( Bifidobacterium animalis subsp. lactis BX-246), Lactobacillus paracasei PC-92 ( Lacticaseibacillus paracasei PC-92) and Lactobacillus rhamnosus Prosci-109 ( Lacticaseibacillus rhamnosus Prosci- 109).

[0023] 2. Main instruments and equipment The main instruments and equipment used in this experiment are shown in Table 1: Table 1 Main instruments and equipment 3. Experimental reagents Quality control microspheres: Mix 1 drop of quality control microspheres with 1 mL of filtered water. Quality control microspheres were purchased from Beckman Coulter (USA).

[0024] Filter membrane: 0.2 μm, purchased from Thermo Fisher Scientific.

[0025] Absolute counting microspheres (Flow-Count Fluorospheres, Beckman): The microspheres have a concentration of 1011 fluorescent particles / mL and are mixed with the stained sample in a 1:1 ratio.

[0026] Sheath fluid: Mix sheath fluid with water in a ratio of 1:7. Filter the prepared sheath fluid in advance and sterilize it in a container at 121°C for 15 minutes. Sheath fluid was purchased from Beckman Coulter (USA).

[0027] MRS medium: Prepare in advance and sterilize. The MRS medium formula is beef extract 10g / L, Tween-80 1g / L, peptone 10g / L, yeast extract 5g / L, glucose 20g / L, potassium dihydrogen phosphate 2g / L, sodium acetate 5g / L, sodium citrate 2g / L, magnesium sulfate heptahydrate 0.2g / L, and manganese sulfate pentahydrate 0.054g / L.

[0028] PBS buffer solution: Filter the solution in advance and add 9 mL to each test tube. Sterilize the solution. The formula for PBS buffer solution is 8 g / L sodium chloride, 1.15 g / L disodium hydrogen phosphate, and 0.2 g / L potassium dihydrogen phosphate. Water is used as the solvent.

[0029] Dyes: 0.5 mM DRAQ7 (Sigma-Aldrich); 1 mM Calcein AM (Thermo Fisher Scientific).

[0030] Centrifuge tube: Use a 2 mL centrifuge tube.

[0031] Pipette and corresponding tips: 100μL (yellow tip), 1mL (blue tip).

[0032] Example 1 1. Strain Activation Activation of the first generation: Inoculate the four strains in the cryopreserved tubes into 10 mL of MRS liquid culture medium and culture them at 37°C in an anaerobic environment for 24 hours. Lacticaseibacillus paracasei PC-01), Bifidobacterium animalis subsp. lactis BX-246 ( Bifidobacterium animalis subsp.lactis BX-246), Lactobacillus paracasei PC-92 ( Lacticaseibacillus paracasei PC-92) and Lactobacillus rhamnosus Prosci-109 ( Lacticaseibacillus rhamnosus Prosci-109).

[0033] Activate the second generation: 1% volume of each of the four bacterial suspensions activated in the first generation was inoculated into 35 mL of MRS liquid culture medium and cultured at 37°C in an anaerobic environment for 24 h.

[0034] Activation of the third generation: The four bacterial suspensions after activation of the second generation were inoculated at a volume fraction of 1% and placed in 2.5 L of MRS liquid culture medium. Each was cultured in an anaerobic environment at 37°C for 24 hours before use.

[0035] The activated third generation bacterial solution of the four strains was centrifuged at 9000 rpm for 15 minutes at 4°C to obtain activated bacterial slurry. The four strains were activated separately to obtain bacterial slurries of different strains.

[0036] 2. Inactivation The activated bacterial sludge of the four strains was inactivated at 95°C for 15 minutes to obtain the inactivated bacterial sludge of different strains, which were the postbiotics of the four strains.

[0037] 3. Vacuum freeze drying Take the inactivated bacterial slurry of each of the four strains and centrifuge it at 9000 rpm and 4°C for 15 minutes. Remove the precipitate and set aside. Wash the precipitate with sterile PBS buffer and remove the precipitate. Repeat this operation three times. Freeze-dry the precipitate at a minimum temperature of -40°C and a vacuum of 15 MPa for 3 hours to obtain bacterial powder. Perform the above operation on each of the four strains to obtain the postbiotic bacterial powder of each strain.

[0038] The postbiotic powder was dried and vacuumed for 30 h, then ground in a mortar and pestle to extract the postbiotic powder and keep it for later use.

[0039] 4. Sample Pretreatment Take 5g of the postbiotic powder of the four strains and place them in a sterile bottle. Add 45mL of PBS buffer solution in a sterile environment to dilute them respectively to obtain sample bacterial suspensions of different strains. Dilute the sample bacterial suspensions of different strains to a concentration of 10 6 CFU / mL~10 7 CFU / mL, set aside.

[0040] 5. Study on dyeing time and dye concentration During sample processing, to facilitate flow cytometric analysis, the sample must be stained and incubated with fluorescent dyes to provide a fluorescent signal that can be collected and processed by the flow cytometer. Therefore, optimizing the fluorescent dye concentration and staining time allows for the most accurate and rapid quantitative analysis of postbiotic cells.

[0041] The present invention uses a single-factor method combined with flow cytometry to test the concentration and staining time of two fluorescent dyes using Lactobacillus paracasei PC-01 bacterial sludge, and the counting unit is cells / g. The specific scheme is:

[0042] Using Lactobacillus paracasei PC-01 as a representative strain, the concentration and staining time of the fluorescent dye were studied. The results of the activated and diluted bacteria were compared with the results of the plate pour method at different concentrations of fluorescent dye and staining time to determine the optimal fluorescent dye concentration and staining time. The specific steps are as follows:

[0043] Plate pouring method: Take 1g of the activated cultured Lactobacillus paracasei PC-01 bacterial sludge and add PBS buffer solution in a sterile environment to dilute it to a bacterial suspension concentration of 10 2 CFU / mL~10 3 CFU / mL. 1 mL of the diluted Lactobacillus paracasei PC-01 suspension was placed into the prepared MRS medium using the pouring method. The culture was placed in an anaerobic environment at 37°C for 24 h. After the colonies grew, they were counted and the number of viable bacteria was calculated.

[0044] The counting result is: (2.10±0.23)×10 12 CFU / g.

[0045] Flow cytometry counting: (1) Take 1g of the activated cultured Lactobacillus paracasei PC-01 and dilute it with 9mL of PBS buffer solution in a sterile environment. Then dilute it six times to a bacterial suspension concentration of 10 6 CFU / mL, set aside.

[0046] (2) Take 2 mL centrifuge tubes, one of which is a negative control (negative), and the other nine are double-stained with Calcein AM / DRAQ7.

[0047] (3) On 10 -6 Use a pipette to draw 980 μL of the bacterial suspension from the gradient and place it into the above 9 centrifuge tubes for calcein AM / PDRAQ7 double staining.

[0048] (4) Negative control (negative): only add bacterial suspension, -5 Use a pipette to draw 980 μL of the bacterial suspension from the gradient and place it into the centrifuge tube mentioned above as a negative control.

[0049] (5) Calcein AM / DRAQ7 double staining: 10 μL of Calcein AM was added to the above centrifuge tube for Calcein AM / DRAQ7 double staining to adjust the final concentrations to 4.0 μM, 5.0 μM, and 6.0 μM, respectively. Then, 10 μL of DRAQ7 was added to the tubes with different concentrations of Calcein AM to adjust the final concentrations to 2.0 μM, 3.0 μM, and 4.0 μM, respectively. Each tube was shaken for 30 seconds. Each sample with different Calcein AM / DRAQ7 double staining concentrations was placed in a 2 mL centrifuge tube and incubated in the dark at room temperature for 30 minutes for staining reaction.

[0050] (6) Take 200 μL of the double-stained samples, add an equal volume of absolute counting microspheres, and shake on an oscillator for 1 minute to mix.

[0051] (7) Flow cytometry was used to count the viable bacteria of Lactobacillus paracasei PC-01. The specific steps were as follows: an unstained sample was used as a negative control, and a FSC-SSC dot plot was established with the forward scatter angle and the side scatter angle as the horizontal and vertical coordinates. A "gate" R5 was set to circle the target cells to be tested. Two fluorescent dyes, DRAQ7 and calcein AM, were used to fluorescently stain dead cells and live cells, respectively. The instrument parameters could be adjusted during the on-machine detection process. A scatter plot was established with the horizontal coordinate of 488-513 / 26-Height-Log and the vertical coordinate of 561-614 / 20-Height-Log. A "gate" R3 was set to circle the number of negative bacteria stained by DRAQ7, i.e., the dead cell population, and a "gate" R4 was set to circle the number of positive bacteria stained by calcein AM, i.e., the live cell population. The scatter plot was used to record the number of dead bacteria, live bacteria, and absolute count microspheres in the sample, and the total bacterial count, its standard deviation, and the average value were calculated. The results are shown in Table 2.

[0052] Table 2 Count results of Lactobacillus paracasei PC-01 at different concentrations of fluorescent dye The concentration and staining time of the fluorescent dye were studied through experiments, and the concentration and staining time corresponding to the result of flow cytometer counting that was closest to the plate counting result and had no significant difference were regarded as the optimal staining conditions.

[0053] As shown in Table 2, the optimal staining concentrations are 5.0 μM for the fluorescent dye Calcein AM and 3.0 μM for DRAQ7. The flow cytometer results are closest to those of the plate pour method and there is no significant difference. The plate pour method results for viable bacteria counts under these staining conditions are: (2.08 ± 0.03) × 10 12 CFU / g.

[0054] The staining time was further optimized under the optimal staining concentration conditions. When the final concentration of fluorescent dye calcein AM was 5.0µM and the final concentration of DRAQ7 was 3.0µM, the flow cytometric counting results at staining time of 15min, 20min, 25min, 30min, and 35min were: (1.62±0.01)×10 12 cells / g, (1.71±0.04)×10 12 cells / g, (2.10±0.11)×10 12 cells / g, (2.03±0.24)×10 12 cells / g, (2.06±0.32)×10 12 cells / g; The above results show that when staining times were 25, 30, and 35 minutes, there was no significant difference between flow cytometry and plate pour count results. Perhaps because the short staining time prevented staining of some bacterial species, the flow cytometry results at 15 and 20 minutes were lower than those at the plate pour count, resulting in a significant difference. However, given that prolonged staining times can lead to issues such as fluorescence quenching, the optimal staining time was ultimately determined to be 25 minutes.

[0055] The above experimental results show that the optimal staining conditions are when the final concentration of the fluorescent dye calcein AM is 5.0µM, the final concentration of DRAQ7 is 3.0µM, and the staining time is 25 minutes.

[0056] 6. Counting Dead Lactic Acid Bacteria Cells in Postbiotics Based on the above optimal staining conditions of a final Calcein AM concentration of 5.0µM and a final DRAQ7 concentration of 3.0µM for 25 minutes, we verified the flow cytometric count results with those obtained by the plate pour method, and found no significant difference, demonstrating the high accuracy of flow cytometry. Next, we used flow cytometry to count dead cells in postbiotics under the above staining conditions.

[0057] The present invention uses Bifidobacterium animalis subspecies lactis BX-246, Lactobacillus paracasei PC-92 and Lactobacillus rhamnosus Prosci-109 in combination with optimized optimal staining conditions to perform method verification, and the method is a flow cytometer counting method.

[0058] The specific steps are as follows: (1) Take four 2 mL centrifuge tubes, one of which is a negative control (negative), and the other three are marked with Calcein AM / DRAQ7 double staining.

[0059] (2) Use a pipette to 6 Pipette 980 μL of the cfu / mL bacterial suspension and place it into 4 centrifuge tubes.

[0060] (3) Negative control (negative): Only bacterial suspension is added to the above centrifuge tube as a negative control.

[0061] (4) Calcein AM / DRAQ7 dye double staining: Add 10 μL of Calcein AM dye with a final concentration of 5.0 μM and 10 μL of DRAQ7 dye with a final concentration of 3.0 μM to three of the centrifuge tubes, shake on a shaker for 30 seconds, and incubate in the dark at room temperature for 25 minutes to obtain double-stained samples.

[0062] (5) Take 200 μL of the double-stained sample, add an equal volume of absolute counting microspheres, and oscillate on an oscillator for 1 minute to mix.

[0063] (6) On-machine testing: After thoroughly mixing the sample and dye, the prepared bacterial sample was analyzed using a flow cytometer. Using an unstained sample as a negative control, an FSC-SSC dot plot was created using forward and side scatter angles as the horizontal and vertical coordinates. Gate R5 was set to identify the target cells.

[0064] Two fluorescent dyes, DRAQ7 and Calcein AM, were used to fluorescently stain dead and live cells, respectively. Instrument parameters can be adjusted during the on-board assay. A scatter plot was created with the horizontal axis 488-513 / 26-Height-Log and the vertical axis 561-614 / 20-Height-Log. A "gate" R was set to delineate the number of DRAQ7-stained negative bacteria, i.e., the dead cell population, and a "gate" R4 was set to delineate the number of Calcein AM-stained positive bacteria, i.e., the live cell population.

[0065] The number of dead bacteria, live bacteria and absolute count microspheres in the sample were recorded using a scatter plot, and the total bacterial count, standard deviation and mean were calculated.

[0066] result: 1. Differentiation of different populations by flow cytometry The division of the colonies was first performed by circling the larger cell colonies using the FSC-SSC negative control graph in the flow cytometric analysis chart. The calcein AM-positive and DRAQ7-negative staining areas were further recorded using the dual fluorescence parameter graph in the flow cytometric analysis chart to circle the dead cell colonies and the live cell colonies. Finally, the bacterial count was calculated using counting microspheres.

[0067] According to the basic principles of flow cytometry, the FSC-SSC diagram in the flow cytometry analysis atlas can analyze the expression of the target cell population, where FSC can determine the size of the cells in the sample and SSC can determine the complexity of the cells.

[0068] 2. Flow cytometry and plate pour count results The stained cells in the postbiotics were counted by flow cytometry analysis, and the number of counting microspheres was recorded to calculate the number of bacteria. The plate pouring counting method was to pour the bacterial liquid into the prepared MRS medium, count the colonies after they were cultured, and record the total number of bacteria.

[0069] The inactivated bacterial sludge was vacuum freeze-dried to obtain bacterial powder. The freeze-drying ratios of the bacterial strains are shown in Table 3. Therefore, the results of plate pour counts under the same conditions were calculated based on the mass ratios of the different bacterial strains and compared with the flow cytometer results. The flow cytometer and plate pour count results are shown in Table 3. The experimental results were analyzed to confirm that flow cytometry can be used to count dead lactic acid bacteria cells in postbiotics, and a method for counting these cells was developed.

[0070] Table 3 Flow cytometry and plate pour count results As shown in Table 3, the plate pour count result of Bifidobacterium animalis subsp. lactis BX-246 was (2.71±0.11)×10 11 CFU / g, the flow cytometer count results before and after inactivation were (1.33±0.01)×10 12 cells / g, (1.62±0.03)×10 12 cells / g, among which the flow cytometric count results after inactivation were higher than those after inactivation, and the flow cytometric count results were higher than those of plate pour counts; the plate pour count result of Lactobacillus paracasei PC-92 was (7.09±0.53)×10 11 CFU / g, the flow cytometer count results before and after inactivation were (1.38±0.44)×10 12 cells / g, (1.36±0.01)×10 12 cells / g, among which the flow cytometric count results after inactivation were lower than those after inactivation and higher than those after plate pouring counts; the plate pouring count result of Lactobacillus rhamnosus Prosci-109 was (1.57±0.49)×10 12 CFU / g, the flow cytometer count results before and after inactivation were (1.27±0.07)×10 12 cells / g, (2.86±0.05)×10 12 cells / g, among which the flow cytometer counting results after inactivation were higher than those after inactivation, and the flow cytometer counting results were higher than the plate pour counting results.

[0071] Sample loss, cell damage, and impurities generated during sample handling can lead to differences in flow cytometer counts before and after inactivation. Flow cytometry counts individual bacteria, while the plate pour count method counts colonies formed by culturable bacteria. Therefore, the results of the two methods are not exactly the same, but the difference is not significant.

[0072] From the above experimental results, it is known that there is no significant difference between the counting results of flow cytometry and plate pour counting. At the same time, flow cytometry is more convenient and faster than the plate pour counting method. Therefore, flow cytometry can be used to count dead lactic acid bacteria cells in postbiotics and develop a quantitative method for cells in postbiotics. Therefore, the present invention provides a method for counting dead lactic acid bacteria cells in postbiotics, which can be used for a method for counting dead lactic acid bacteria cells in postbiotics, and the method comprises the following steps:

[0073] (1) Sample pretreatment The strain to be tested was activated, and the activated third-generation bacterial solution was centrifuged at 9000 rpm for 15 minutes at 4°C to obtain a bacterial slurry of the strain to be tested. The activated bacterial slurry of the strain to be tested was inactivated at 95°C for 15 minutes to obtain an inactivated bacterial slurry of the strain to be tested. The inactivated bacterial slurry of the strain to be tested was centrifuged at 9000 rpm at 4°C for 25 minutes, and the precipitate was removed for later use. The precipitate was washed with sterile PBS buffer and removed. This operation was repeated three times. The precipitate was freeze-dried at a minimum temperature of -40°C and a vacuum of 15 MPa. After pre-freezing for 3 hours, the postbiotic powder was obtained.

[0074] Take 5g of postbiotic powder and place it in a sterile bottle. Add 45mL of PBS buffer solution in a sterile environment to dilute the sample bacterial suspension to a concentration of 10 6 CFU / mL~10 7 CFU / mL, and obtain the bacterial suspension of the sample to be tested.

[0075] (2) Flow cytometry counting.

[0076] (3) Take two 2 mL centrifuge tubes, one of which is a negative control (negative), and the other is for Calcein AM / DRAQ7 double staining.

[0077] (4) At a concentration of 10 6 Use a pipette to draw 980 μL of the CFU / mL bacterial suspension and place it in a centrifuge tube.

[0078] (5) Negative control (negative): only add bacterial suspension.

[0079] (6) Calcein AM / DRAQ7 double staining: Add 10 μL of Calcein AM (final concentration 5.0 μM) and 10 μL of DRAQ7 (final concentration 3.0 μM) to the sample and shake for 30 seconds. Incubate in the dark at room temperature for 25 minutes to obtain a double-stained sample. Take 200 μL of the double-stained sample and add an equal volume of absolute counting microspheres. Shake on a shaker for 1 minute to mix.

[0080] (7) On-machine testing After thoroughly mixing the sample and dye, the prepared bacterial sample was analyzed using a flow cytometer. Using an unstained sample as a negative control, an FSC-SSC dot plot was created using forward and side scatter angles as the horizontal and vertical coordinates. Gate R5 was set to identify the target cells.

[0081] Two fluorescent dyes, DRAQ7 and Calcein AM, were used to fluorescently stain dead and live cells, respectively. Instrument parameters can be adjusted during the on-board assay. A scatter plot was created with the horizontal axis 488-513 / 26-Height-Log and the vertical axis 561-614 / 20-Height-Log. A "gate" R was set to delineate the number of DRAQ7-stained negative bacteria, i.e., the dead cell population, and a "gate" R4 was set to delineate the number of Calcein AM-stained positive bacteria, i.e., the live cell population.

[0082] The number of dead bacteria, live bacteria and absolute count microspheres in the sample were recorded using a scatter plot, and the total bacterial count, standard deviation and mean were calculated.

[0083] This invention provides a method for counting dead lactic acid bacteria cells in postbiotics, which is a convenient and rapid method for quantifying cells in postbiotics. The method uses postbiotics with probiotic properties screened in previous laboratory studies as research subjects. Through orthogonal experiments, the method optimizes the fluorescent dye staining time and concentration combination to determine the optimal staining time and concentration combination. Flow cytometry is then used to count dead lactic acid bacteria cells in the postbiotics. The results are compared with those from traditional plate pour counts, resulting in a more convenient and rapid method for counting dead lactic acid bacteria cells in postbiotics.

[0084] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0085] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0086] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for counting dead lactic acid bacteria cells in postbiotics, characterized in that: The steps include: Sample pretreatment: Take the postbiotic powder to be tested and prepare the sample bacterial suspension; Flow cytometry counting: dilute the sample bacterial suspension to 1.0×10 6 CFU / mL~1.0×10 7 CFU / mL, two fluorescent dyes are added, and the mixture is incubated in the dark at room temperature to obtain a double-stained sample; the double-stained sample is taken and quantitative analysis and detection is performed using a flow cytometer to obtain the number of dead lactic acid bacteria cells in the postbiotic; wherein the fluorescent dyes include DRAQ7 and calcein AM; the final concentration of the DRAQ7 fluorescent dye is 2.8µM~3.2µM, and the final concentration of the calcein AM is 4.8µM~5.0µM.

2. The method for counting dead lactic acid bacteria cells in a postbiotic according to claim 1, characterized in that: The incubation time at room temperature in the dark is 20 min to 30 min.

3. The method for counting dead lactic acid bacteria cells in a postbiotic according to claim 1, characterized in that: The preparation method of the sample bacterial suspension is as follows: Take the postbiotic bacterial powder to be tested, add a diluent to dilute it under a sterile environment, and obtain the sample bacterial suspension.

4. The method for counting dead lactic acid bacteria cells in a postbiotic according to claim 3, characterized in that: The diluent is a PBS buffer solution; the PBS buffer solution is composed of the following effective substances at the following final concentrations: Sodium chloride 7.8g / L~8.2g / L, disodium hydrogen phosphate 1.13g / L~1.17g / L and potassium dihydrogen phosphate 0.1g / L~0.3g / L, the solvent is water.

5. The method for counting dead lactic acid bacteria cells in a postbiotic according to claim 3, characterized in that: The postbiotic bacterial powder to be tested is obtained by inactivating the strain to be tested and then subjecting it to vacuum freeze-drying.

6. The method for counting dead lactic acid bacteria cells in a postbiotic according to claim 5, characterized in that: The strain to be tested includes at least one of Lactobacillus paracasei PC-01, Bifidobacterium animalis subspecies lactis BX-246, Lactobacillus paracasei PC-92 and Lactobacillus rhamnosus Prosci-109.

7. The method for counting dead lactic acid bacteria cells in a postbiotic according to claim 5, characterized in that: The inactivation conditions are 94° C. to 96° C. for 20 min to 30 min.

8. The method for counting dead lactic acid bacteria cells in postbiotics according to claim 5, characterized in that: The vacuum freeze-drying conditions are as follows: a minimum temperature of -35°C to 45°C, a vacuum degree of 14 MPa to 15 MPa, and a pre-freezing time of 2.8 hours to 3.2 hours.

9. The method for counting dead lactic acid bacteria cells in a postbiotic according to claim 1, characterized in that: Before quantitative analysis and detection using flow cytometry, an equal volume of absolute counting microspheres was added to the double-stained sample and mixed.

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