A method for rapid detection of live / dead bacteria in animal Bifidobacterium lactis subsp.

Abstract

The present invention relates to a method for rapid detection of live / dead bacteria in animal Bifidobacterium lactis subspecies fermentation liquid, the method comprising: centrifuging the fermentation liquid and discarding the supernatant to obtain a bacterial precipitate, diluting to obtain a bacterial suspension, mixing with SYTO 9 / PI fluorescent dye, incubating, and analyzing by flow cytometry. The present invention detects cells after SYTO 9 and PI double staining by flow cytometry, can effectively distinguish live bacteria (green fluorescence) from dead bacteria (red fluorescence), achieves high-throughput distinction between live bacteria and dead bacteria, is used for rapid and accurate determination of live / dead bacteria counts of animal Bifidobacterium lactis subspecies, improves detection efficiency and accuracy, and provides strong technical support for quality control of probiotic fermentation products.

Description

Technical Field

[0001] The invention relates to the field of microbial detection, and in particular to a method for rapid detection of live / dead bacteria in animal Bifidobacterium lactis subspecies fermentation liquid. Background Art

[0002] In recent years, the application of probiotics in the field of food and health has received extensive attention. Among them, bifidobacterium, as an important probiotic, has received a lot of research and attention from the industry because of its significant role in improving intestinal microecology, enhancing immune function and promoting nutrient absorption. Animal Bifidobacterium lactis subspecies (Bifidobacterium animalissubsp.lactis) is a probiotic commonly used in dairy fermentation, with good intestinal colonization ability and immunomodulatory function. Studies have shown that animal Bifidobacterium lactis subspecies has significant effects in improving digestive health, enhancing immunity, and preventing and treating diarrhea. How to accurately determine the activity and quantity of animal Bifidobacterium lactis subspecies during the production process is the key to ensuring product quality and benefits. In addition, in the fermentation production of probiotics, controlling the activity and quantity of strains, and how to accurately and efficiently detect the ratio of live bacteria to dead bacteria are important links to ensure product efficacy. Traditional detection methods such as plate counting methods are time-consuming and have limited accuracy, and cannot meet the rapid detection needs of modern fermentation industry.

[0003] CN110055301A discloses a method for rapid detection and counting of bifidobacteria. The culture medium used for detecting bifidobacteria includes a basic culture medium, a chromogenic substrate 5-bromo-4-chloro-3-indole-α-D-galactoside and / or 5-bromo-4-chloro-3-indole-β-D-galactoside. During the culture process of bifidobacteria, α-galactosidase is produced by metabolism and reacts with the chromogenic substrate to release indole, so that blue bifidobacterium colonies appear on the culture medium or a blue halo is produced around the bifidobacterium colonies. Interfering bacteria cannot show blue color without α-galactosidase, and the chromogenic substrate has an inhibitory effect on other interfering bacteria. By adding a surfactant, the colony is facilitated to grow rapidly; antibiotics are added to the culture medium for detecting bifidobacteria to inhibit the growth of other interfering bacteria.

[0004] CN101319250A discloses a method for rapid quantitative detection of live bifidobacteria in bifidobacterium-containing products. The method comprises the following steps: taking a sample, diluting the sample 10 times, performing EMA treatment, extracting DNA from the sample, performing molecular beacon-real-time PCR detection, and calculating the content of bifidobacteria according to a standard curve. The DNA extraction process is as follows: adding 18% sodium citrate and 1M NaOH to the sample after EMA treatment, centrifuging at 10000 rpm for 10 minutes, collecting bacterial cells, washing with ultrapure water, taking a bacterial suspension, adding 2% Tritox-100, treating the sample in a 100°C water bath for 10 minutes, and immediately cooling the supernatant for molecular beacon-real-time PCR amplification.

[0005] Flow cytometry is an efficient cell analysis technique that can quickly and quantitatively analyze a large number of single cells by measuring the scattered light and fluorescence generated when the cell suspension flows through the laser beam. The SYTO 9 and propidium iodide (PI) double staining technique is an important method to distinguish live bacteria from dead bacteria. SYTO 9 is a green fluorescent dye that can penetrate all cell membranes and bind to nucleic acids, while PI can only penetrate damaged cell membranes and emit red fluorescence after binding to nucleic acids.

[0006] In summary, how to develop a rapid, accurate and high-throughput determination method for the number of live and dead bacteria of Bifidobacterium lactis subsp. animalis has become one of the urgent problems to be solved in this field. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for rapid detection of live / dead bacteria in animal Bifidobacterium lactis fermentation broth. By detecting cells double-stained with SYTO 9 and PI by flow cytometer, live bacteria (green fluorescence) and dead bacteria (red fluorescence) can be effectively distinguished, thereby achieving high-throughput distinction between live and dead bacteria, and being used for rapid and accurate determination of the number of live / dead bacteria in animal Bifidobacterium lactis.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for rapid detection of live / dead bacteria in a fermentation broth of animal Bifidobacterium lactis subsp. lactis, the method comprising: centrifuging the fermentation broth to discard the supernatant to obtain a bacterial precipitate, diluting the bacterial suspension to obtain a bacterial suspension, mixing the suspension with SYTO 9 / PI fluorescent dye, incubating, and analyzing the suspension by flow cytometry.

[0010] The present invention detects cells after SYTO 9 and PI double staining by flow cytometry, can effectively distinguish live bacteria (green fluorescence) and dead bacteria (red fluorescence), realizes high-throughput distinction between live bacteria and dead bacteria, is used for rapid and accurate determination of live / dead bacteria counts of animal Bifidobacterium lactis subspecies, improves detection efficiency and accuracy, and provides strong technical support for quality control of probiotic fermentation products. Compared with the traditional plate counting method, not only the number of live bacteria can be detected, but also the number of dead bacteria can be detected, and has the characteristics of high efficiency and high accuracy.

[0011] Preferably, the centrifugation and discarding the supernatant specifically comprises: discarding the supernatant of the fermentation broth after a first centrifugation, resuspending the fermentation broth in a diluent, and collecting the precipitate by a second centrifugation.

[0012] Preferably, the temperatures of the first centrifugation and the second centrifugation are independently 0-8°C (for example, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, etc.).

[0013] Preferably, the rotation speeds of the first centrifugation and the second centrifugation are independently 5000-10000 r / min (for example, 5000 r / min, 6000 r / min, 7000 r / min, 8000 r / min, 9000 r / min or 10000 r / min, etc.).

[0014] Preferably, the time for the first centrifugation and the second centrifugation is independently 2 to 8 minutes (for example, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes or 8 minutes, etc.).

[0015] Preferably, the dilution comprises: using a diluent to dilute the bacterial precipitate until the number of bacterial cells is 5×10 5 ~1×10 7 CFU / mL (e.g., 5×10 5 CFU / mL, 8×10 5 CFU / mL, 1×10 6 CFU / mL, 2×10 6 CFU / mL, 5×10 6 CFU / mL, 7×10 6 CFU / mL or 1×10 7 CFU / mL, etc.).

[0016] Preferably, the diluent contains a PBS buffer having a pH of 7 to 8 (eg, 7, 7.2, 7.4, 7.6, 7.8 or 8, etc.).

[0017] Preferably, the volume ratio of the bacterial suspension to the SYTO 9 / PI fluorescent dye is (500-1500):1.

[0018] The specific point values ​​of 500 to 1500 mentioned above may be 500, 700, 900, 1000, 1100, 1300 or 1500 etc.

[0019] Preferably, the SYTO 9 / PI fluorescent dye comprises SYTO 9 dye and PI dye.

[0020] Preferably, in the SYTO 9 / PI fluorescent dye, the volume ratio of SYTO 9 dye to PI dye is (3-5):1.

[0021] The specific point values ​​of 3 to 5 mentioned above can be selected from 3, 3.5, 3.8, 4, 4.2, 4.5 or 5, etc.

[0022] Preferably, the preparation method of the SYTO 9 / PI fluorescent dye comprises: mixing SYTO 9 dye with a concentration of 3.34 mM and PI dye with a concentration of 20 mM in a volume ratio of (3-5):1.

[0023] Preferably, the incubation is light-proof incubation.

[0024] Preferably, the incubation time is 20 to 30 min (eg, 20, 23, 25, 27 or 30, etc.).

[0025] Preferably, the incubation temperature is 20-30°C (for example, 20°C, 23°C, 25°C, 27°C or 30°C, etc.).

[0026] Preferably, the excitation wavelength of the flow cytometer analysis is 488 nm, and the fluorescence channels are B525 and B585.

[0027] Preferably, the setting parameters of the flow cytometer include: a total number of particles read is 20,000, a FSC channel gain is 1,000, a SSC channel gain is 1,000, a B525 channel gain is 30, and a B585 channel gain is 80.

[0028] Preferably, the threshold values ​​of the flow cytometer are set to: FSC 50000, SSC 10000.

[0029] Preferably, the compensation settings of the flow cytometer are: B525 6%, B585 64%.

[0030] Preferably, the method comprises the following steps:

[0031] (1) Sample preparation: The fermented liquid of Bifidobacterium animalis subsp. lactis was centrifuged at 5000-10000 r / min for 2-8 min at 0-8°C, the supernatant was discarded, the liquid was resuspended in PBS buffer with a pH of 7-8, and the liquid was centrifuged at 5000-10000 r / min for 2-8 min at 0-8°C, the precipitate was collected, and the bacterial precipitate was diluted with PBS buffer with a pH of 7-8 until the bacterial count was 5×10 5 ~1×10 7 CFU / mL, and bacterial suspension was obtained;

[0032] (2) Fluorescent staining: mixing the bacterial suspension with SYTO 9 / PI fluorescent dye, incubating at 20-30° C. in the dark for 20-30 min, and performing SYTO 9 / PI fluorescent staining, wherein the volume ratio of the bacterial suspension to the SYTO 9 / PI fluorescent dye is (500-1500):1, the SYTO 9 / PI fluorescent dye comprises SYTO 9 dye and PI dye, and the volume ratio of the SYTO 9 dye to the PI dye is (3-5):1. The preparation method of the SYTO 9 / PI fluorescent dye comprises mixing SYTO 9 dye with a concentration of 3.34 mM and PI dye with a concentration of 20 mM at a volume ratio of (3-5):1;

[0033] (3) Flow cytometry: After incubation, the cells were analyzed by flow cytometry immediately with an excitation wavelength of 488 nm, fluorescence channels of B525 and B585, a total number of particles read of 20,000, FSC channel gain of 1,000, SSC channel gain of 1,000, B525 channel gain of 30, B585 channel gain of 80, thresholds set to FSC 50,000, SSC 10,000, and compensations set to B525 6% and B585 64%.

[0034] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The invention provides a detection method based on flow cytometry and SYTO 9 / PI double staining technology. By detecting cells after SYTO 9 and PI double staining by flow cytometer, live bacteria (green fluorescence) and dead bacteria (red fluorescence) can be effectively distinguished, and high-throughput distinction between live bacteria and dead bacteria is achieved. It is used for rapid and accurate determination of live / dead bacteria counts of animal Bifidobacterium lactis subspecies, improves detection efficiency and accuracy, and provides strong technical support for quality control of probiotic fermentation products. Compared with the traditional plate counting method, it can detect not only the number of live bacteria but also the number of dead bacteria, and has the characteristics of high efficiency and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The gated dot plots for live / dead cells in flow cytometry are shown in Figure A, where Figure A is the FSC-SSC dual-parameter scatter plot of BLa80 cells, Figure B is the SYTO 9 single staining image of BLa80 cells, Figure C is the PI single staining image of BLa80 cells, and Figure D is the SYTO9 / PI double staining image of BLa80 cells.

[0038] Figure 2 This is a test result diagram of Example 1 in Test Example 1.

[0039] Figure 3 This is a graph showing the OD value of BLa80 fermentation broth changing over time at different growth stages.

[0040] Figure 4 This is a graph showing the pH value of BLa80 fermentation broth changing over time at different growth stages.

[0041] Figure 5 This is a graph showing the results of determining the number of viable bacteria in BLa80 fermentation broth using flow cytometry and plate counting at different growth stages. DETAILED DESCRIPTION

[0042] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0043] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0044] The culture medium involved in the following examples is as follows:

[0045] MRS medium (g / L): Weigh 10 g of peptone, 10 g of beef extract, 15 g of glucose, 15 g of lactose, 5 g of yeast powder, 2 g of diammonium hydrogen citrate, 2.6 g of K2PO4·3H2O, 0.1 g of MgSO4·7H2O, 0.05 g of MnSO4, 1 mL of Tween 80, and 0.5 g of cysteine ​​salt, add them to 1 L of deionized water, stir thoroughly to dissolve, adjust the pH to 6.5, and sterilize under high pressure at 121°C for 20 min.

[0046] PBS buffer: weigh 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, and 0.27 g KH2PO4 and add them to 1 L deionized water. Stir thoroughly to dissolve, then add concentrated hydrochloric acid or NaOH to adjust the pH. Sterilize at 121°C under high pressure for 20 min.

[0047] The BLa80 strain involved in the following embodiment is classified and named as Bifidobacterium animalis subsp. lactis, and the preservation unit is the General Microbiology Center of China Culture Collection Administration, the preservation time is May 17, 2021, the preservation number is CGMCC No. 22547, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0048] The BLa36 strain involved in the following embodiments is classified and named as Bifidobacterium animalis subsp. lactis, and the preservation unit is the General Microbiology Center of China Culture Collection Administration. The preservation time is December 2, 2021, the preservation number is CGMCC No. 24029, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0049] Example 1

[0050] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium lactis subsp. animalis, comprising the following steps:

[0051] (1) The BLa80 strain was inoculated into MRS liquid medium and cultured at 37°C for 24 hours to activate the culture medium, and then the activated solution was inoculated into MRS liquid medium at an inoculum volume of 2% (v / v), and then cultured at 37°C for 16 hours to obtain a bacterial solution. The bacterial solution was centrifuged at 8000 r / min for 5 minutes at 4°C, the supernatant was discarded, and the solution was resuspended in PBS with a pH of 7.4, and then centrifuged at 8000 r / min for 5 minutes at 4°C, and the supernatant was discarded. The bacterial cells were resuspended in PBS with a pH of 7.4 and diluted to about 2×10 6 CFU / mL concentration to obtain BLa80 bacterial suspension.

[0052] (2) Take two tubes of bacterial suspension prepared in step (1), boil one tube in a 100°C water bath for 10 min to inactivate all the bacteria, and then add an equal volume of untreated live bacterial suspension to prepare a bacterial suspension with a live-to-dead ratio of 1:1.

[0053] (3) Take 0.5 mL of the bacterial suspension of step (2), mix it with the double dye, the volume ratio of the bacterial suspension to the double dye is 1000:1, and incubate at 25°C in the dark for 25 minutes. The initial concentration of SYTO 9 dye is 3.34 mM, the initial concentration of PI dye is 20 mM, and SYTO 9 dye and PI dye are mixed at a volume ratio of 4:1 to prepare the double dye.

[0054] (4) Gain and compensation adjustment:

[0055] ① Select the bacterial suspension of step (1) as a blank control. By adjusting the photodiode voltage of the flow cytometer to adjust the signal intensity of the forward scattered light FSC and the side scattered light SSC, the FSC / SSC gate is set to assist in determining the position of the cells to be tested in the two-dimensional dot plot of the flow cytometer to eliminate the autofluorescence of the strain to be tested, and the working voltages of FSC and SSC are both 1000. The scatter plot results are as follows: Figure 1 As shown in Figure A.

[0056] ②SYTO 9 single staining as a live cell gating control. The steps are: add 0.4μL SYTO 9 dye to the blank control bacterial solution in step ①, the concentration of SYTO 9 dye is 3.34mM, shake and mix, incubate at 25℃ in the dark for 25min, then detect on a flow cytometer, set the gate with the SSC / FSC set in step ①, adjust the PMT voltage of SYTO 9 for gain adjustment, and determine that the voltage of SYTO 9 fluorescence intensity is 30. The scatter plot results are as follows: Figure 1 As shown in Figure B.

[0057] ③PI single staining as a dead cell gating control. The steps are: add 0.1μL PI dye to the bacterial suspension in step (2), the concentration of PI dye is 20mM, shake and mix, incubate at 25℃ in the dark for 25min, then detect on a flow cytometer, set the gate with the SSC / FSC set in step ①, adjust the PMT voltage of PI for gain adjustment, and determine that the voltage of PI fluorescence intensity is 80. The scatter plot results are as follows: Figure 1 As shown in Figure C.

[0058] ④SYTO 9 and PI double staining, determine voltage and compensation. The steps are: add 0.4μL SYTO 9 and 0.1μL PI dyes to the blank control bacterial solution in step ①, shake and mix, incubate at 25℃ in the dark for 25min; then detect on the flow cytometer, set the FSC / SSC gate set in step ①, adjust the PMT voltage of SYTO 9 and PI for dual-color fluorescence compensation, determine the voltage of SYTO9 and PI fluorescence intensity to 30 and 80 respectively, and set the compensation to B525 6% and B585 64%. The scatter plot results are as follows Figure 1 As shown in Figure D.

[0059] In summary, the analysis parameters of flow cytometry are shown in Table 1.

[0060] Table 1

[0061] FSC SSC B525 B585 Gain 1000 1000 30 80 Threshold 50000 10000 / / compensate / / 6％ 64％

[0062] (5) Take the stained bacterial suspension obtained in step (3), set the flow cytometry test parameters according to step (4), and perform the test on the flow cytometer.

[0063] Example 2

[0064] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium lactis subsp. animalis, comprising the following steps:

[0065] (1) The BLa80 strain was inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours to activate the culture medium, and then the activated solution was inoculated into MRS liquid culture medium at an inoculum rate of 2% (v / v), and cultured at 37°C for 16 hours to obtain a bacterial solution. The bacterial solution was centrifuged at 5000 r / min for 8 minutes at 0°C, the supernatant was discarded, and the solution was resuspended in PBS with a pH of 7, and then centrifuged at 10000 r / min for 2 minutes at 8°C, and the supernatant was discarded. The bacterial cells were resuspended in PBS with a pH of 7 and diluted to about 5×10 5 CFU / mL concentration to obtain BLa80 bacterial suspension.

[0066] (2) Take two tubes of bacterial suspension prepared in step (1), boil one tube in a 100°C water bath for 10 min to inactivate all the bacteria, and then add an equal volume of untreated live bacterial suspension to prepare a bacterial suspension with a live-to-dead ratio of 1:1.

[0067] (3) Take 0.5 mL of bacterial suspension and mix it with the double dye, the volume ratio of bacterial suspension to double dye is 1500:1, and incubate at 20°C in the dark for 30 minutes. Among them, the initial concentration of SYTO 9 dye is 3.34 mM, the initial concentration of PI dye is 20 mM, and SYTO 9 dye and PI dye are mixed at a volume ratio of 3:1 to prepare the double dye.

[0068] (4) Take the stained bacterial suspension obtained in step (3), set the flow cytometry test parameters according to Table 1, and perform the test on the machine.

[0069] Example 3

[0070] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium lactis subsp. animalis, comprising the following steps:

[0071] (1) The BLa80 strain was inoculated into MRS liquid culture medium and cultured at 37°C for 24 hours to activate the culture medium to obtain an activated solution; the activated solution was inoculated into MRS liquid culture medium at an inoculum volume of 2% (v / v) and cultured at 37°C for 16 hours to obtain a bacterial solution; the bacterial solution was centrifuged at 10,000 r / min at 8°C for 2 minutes, the supernatant was discarded, and the solution was resuspended in PBS with a pH of 8, and then centrifuged at 5,000 r / min at 0°C for 8 minutes, and the supernatant was discarded. The bacteria were resuspended in PBS with a pH of 8 and diluted to about 1×10 7 CFU / mL concentration to obtain BLa80 bacterial suspension.

[0072] (2) Take two tubes of bacterial suspension prepared in step (1), boil one tube in a 100°C water bath for 10 min to inactivate all the bacteria, and then add an equal volume of untreated live bacterial suspension to prepare a bacterial suspension with a live-to-dead ratio of 1:1.

[0073] (3) Take 0.5 mL of bacterial suspension and mix it with the double dye, the volume ratio of bacterial suspension to double dye is 500:1, and incubate at 30°C in the dark for 20 minutes. The initial concentration of SYTO 9 dye is 3.34 mM, the initial concentration of PI dye is 20 mM, and SYTO 9 dye and PI dye are mixed at a volume ratio of 5:1 to prepare the double dye.

[0074] (4) Take the stained bacterial suspension obtained in step (3), set the flow cytometry test parameters according to Table 1, and perform the test on the machine.

[0075] Example 4

[0076] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of animal Bifidobacterium lactis subsp. lactis. The only difference from Embodiment 1 is that the volume ratio of SYTO 9 dye and PI dye in the double dye in step (3) is 3:1.

[0077] Example 5

[0078] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium animalis subsp. lactis. The only difference from Embodiment 1 is that the volume ratio of SYTO 9 dye and PI dye in the double dye in step (3) is 5:1.

[0079] Example 6

[0080] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium animalis subsp. lactis. The only difference from Embodiment 1 is that the volume ratio of SYTO 9 dye and PI dye in the double dye in step (3) is 2:1.

[0081] Example 7

[0082] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium animalis subsp. lactis. The only difference from Embodiment 1 is that the volume ratio of SYTO 9 dye and PI dye in the double dye in step (3) is 6:1.

[0083] Example 8

[0084] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium lactis subsp. animalis. The only difference from Embodiment 1 is that the incubation time in step (3) is 20 min.

[0085] Example 9

[0086] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium lactis subsp. animalis. The only difference from Embodiment 1 is that the incubation time in step (3) is 30 min.

[0087] Example 10

[0088] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium animalis subsp. lactis. The only difference from Embodiment 1 is that the incubation time in step (3) is 15 min.

[0089] Embodiment 11

[0090] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium lactis subsp. animalis. The only difference from Embodiment 1 is that the incubation time in step (3) is 35 min.

[0091] Example 12

[0092] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium animalis subsp. lactis. The difference from Embodiment 1 is that the PBS buffer used in collecting and diluting the bacteria in step (1) is replaced with an equal amount of sterile water.

[0093] Embodiment 13

[0094] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium animalis subsp. lactis. The difference from Embodiment 1 is that the PBS buffer used in collecting and diluting the bacteria in step (1) is replaced with an equal amount of Hepes buffer of the same pH.

[0095] Embodiment 14

[0096] This embodiment provides a method for rapid detection of live / dead bacteria in a fermentation broth of Bifidobacterium animalis subsp. lactis, which is different from Embodiment 1 only in that the strain is BLa36.

[0097] Embodiment 15

[0098] This embodiment provides a method for rapid detection of live / dead bacteria in fermentation broth of Bifidobacterium animalis subsp. lactis, which is different from Embodiment 1 only in that the strain is Bifidobacterium animalis subsp. lactis ATCC700541.

[0099] Comparative Example 1

[0100] The Lp05 strain involved in this comparative example is classified and named Lactobacillus plantarum. The preservation unit is the General Microbiology Center of China Culture Collection Administration. The preservation time is October 9, 2021. The preservation number is CGMCC No. 23547. The preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0101] This comparative example provides a method for rapid detection of live / dead bacteria in Lactobacillus plantarum fermentation broth, which differs from Example 1 only in that the strain is Lp05.

[0102] Test Example 1

[0103] This test example uses the methods provided in Examples 1-7 and 12-13 to detect the live bacteria ratio and dead bacteria ratio of the same BLa80 strain fermentation broth. The closer the live bacteria to dead bacteria ratio is to 1:1, the more accurate the detection method provided in this example is. The test results are shown in Table 2. The flow cytometry results of Example 1 are shown in Table 2. Figure 2 shown.

[0104] Table 2

[0105]

[0106]

[0107] From Table 2 we can see that:

[0108] (1) Comparing Example 1 with Examples 4-7, it can be seen that under the same cell concentration, after incubation for 25 minutes, when the volume ratio of SYTO 9 and PI is 3:1, 4:1 or 5:1, they have similar proportions of live and dead bacteria; when the volume ratio of SYTO 9 and PI is 2:1, the PI dye accounts for a large proportion in the mixed dye system, so the proportion of dead bacteria is high; when the volume ratio of SYTO 9 and PI is 6:1, the SYTO 9 dye accounts for a large proportion in the mixed dye system, so the proportion of live bacteria is high. In summary, the volume ratio of SYTO 9 and PI is (3-5) is the best ratio, preferably a volume ratio of 4:1.

[0109] (2) Comparing Example 1 with Examples 12-13, it can be seen that the type of diluent will affect the accuracy of the test results. When a diluent other than PBS buffer is used, that is, when the PBS buffer used is replaced with an equal amount of sterile water, the proportions of live bacteria and dead bacteria are 59.34% and 29.76%, respectively; when the PBS buffer used is replaced with an equal amount of Hepes buffer with the same pH, the proportions of live bacteria and dead bacteria are 73.65% and 16.31%, respectively, both of which are significantly different from the theoretical value of 1:1 for live and dead bacteria. In summary, compared with sterile water and Hepes buffer, PBS buffer has the best detection effect.

[0110] Test Example 2

[0111] This test example uses the method provided in Examples 1 and 8-11 to detect the number of viable bacteria in the fermentation broth of the same BLa80 strain, and uses the plate count method. The experimental steps of the plate count method include: diluting the sample solution taken out in step (1) of Examples 1 and 8-11 by 10 times to 10 -3 , 10 -4 , 10 -5 Three gradients of bacterial solution were coated on MRS agar plates, with 3 parallels for each gradient. After anaerobic culture at 37°C for 48 hours, the number of viable bacteria on the plates was recorded. The results obtained by the plate count method were used as the standard. The smaller the relative error, the higher the accuracy. The test results are shown in Table 3.

[0112] Table 3

[0113]

[0114] As shown in Table 3, at different incubation times, the flow cytometer counting results were higher than the plate counting results. Specifically, in Example 1, the incubation time was 25 min, and the flow cytometer counting result was 1728×10 3 AFU / μL, the relative error compared to the plate count result is 4.10%, which is closer to the plate result and has high stability. Comparing Example 1 with Example 8 and Example 9, it can be seen that when the incubation time is 20 to 30 minutes, better flow cytometry results can be obtained, preferably 25 minutes. Comparing Example 1 with Example 10, it can be seen that the flow cytometer count result is the highest when the incubation time is 15 minutes, reaching 1779×10 3 AFU / μL, compared with the plate count result of 1660×10 3 The relative error of CFU / μL was 7.17%, and the flow counting result was unstable, with large differences in the values ​​within the group. Comparing Example 1 with Example 11, it can be seen that after 30 minutes of incubation, the flow counting result tends to be stable as the incubation time increases, so the incubation time of 20 to 30 minutes is selected as the optimal incubation time.

[0115] Test Example 3

[0116] This test example uses the method provided in Example 14, Example 15 and Comparative Example 1 to detect the number of viable bacteria in the fermentation broth, and adopts the plate count method. The experimental steps of the plate count method include: diluting the sample solution taken out in step (1) of Example 14, Example 15 and Comparative Example 1 by 10 times to 10 -3 , 10 -4 , 10 -5 Three gradients of bacterial solution were coated on MRS agar plates, with 3 parallels for each gradient. After anaerobic culture at 37°C for 48 hours, the number of viable bacteria on the plates was recorded. The results obtained by the plate count method were used as the standard. The smaller the relative error, the higher the accuracy. The test results are shown in Table 4.

[0117] Table 4

[0118]

[0119] As shown in Table 4, the flow cytometer count results were close to the plate count results when the detection methods provided in Examples 14 and 15 were used to detect other strains of animal Bifidobacterium lactis subspecies level. Specifically, Example 14 is the count result of detecting BLa36, in which the flow cytometer count result was 2711×10 3 AFU / μL, the relative error compared to the plate counting result was 4.15%; Example 15 is the counting result of ATCC700541, in which the flow counting result was 1415×10 3 AFU / μL, the relative error compared to the plate count result is 4.58%, therefore, the above results can verify the general applicability of the flow cytometer counting method to animal Bifidobacterium lactis subspecies strains. However, the detection object in Comparative Example 1 is Lactobacillus plantarum, and the flow counting result is different from the plate counting result. Therefore, the detection method provided by the present invention cannot be applied to Lactobacillus plantarum, but is a detection method applicable to animal Bifidobacterium lactis subspecies.

[0120] Test Example 4

[0121] This test case applies flow cytometry to fermentation monitoring of the BLa80 strain, including the following steps:

[0122] (1) Inoculate the BLa80 seed solution at a ratio of 2% (v / v) into a sterile anaerobic fermentation tube containing 1.5 L of fermentation culture. Set the fermentation conditions: 37°C, anaerobic culture for 24 hours.

[0123] (2) Starting from the start of the anaerobic fermentation tube operation, samples were taken every 4 hours to measure the OD value, pH value and viable bacterial count of BLa80 at different fermentation stages (using flow cytometry and plate count).

[0124] (3) The OD value determination method is as follows: fully mix the bacterial solution to be tested, draw the sample solution in 1 mL of the test tube, add 4 mL of 0.2% NaOH, mix thoroughly with a vortex oscillator, and prepare a 1:5 sample homogenate for later use. Turn on the power switch of the UV spectrophotometer, preheat for 30 minutes, adjust the wavelength to 600 nm and calibrate, draw about 1 mL of sample, slowly add it to the cuvette until it is 2 / 3 of the cuvette volume, detect on the machine, read and record the OD value.

[0125] (4) The pH value is determined as follows: turn on the power switch of the pH meter for calibration, then fully mix the bacterial solution to be tested, slowly insert the pH probe into the fermentation solution, and read and record the pH value after the pH value on the display screen stabilizes.

[0126] (5) Perform flow cytometry detection according to the method provided in Example 1.

[0127] (6) Perform plate counting simultaneously according to the method provided in Test Example 2.

[0128] Depend on Figure 3 It can be seen that BLa80 entered the logarithmic growth phase after 4 hours of culture, and the OD600 reached a maximum of 2.415 at 16 hours of culture, and then entered the stable phase. Figure 4 It can be seen that the pH value of BLa80 decreased rapidly from 5.72 to 4.66 during 4-12 hours, and then the pH of the fermentation broth decreased slowly, reaching 4.39 after 24 hours.

[0129] The BLa80 fermentation broth at different culture times was detected by flow cytometry and plate counting method. Figure 5 It can be seen that BLa80 was in the logarithmic growth phase at 8 h of culture, and the number of viable bacteria increased slowly after 12 h of culture. The number of viable bacteria reached the maximum at 20 h, that is, the flow count was 2.08×10 9 AFU / μL, plate count was 1.72×10 9 CFU / μL, and then the number of viable bacteria decreased rapidly. Comparison of the differences in the counting results of flow cytometry and plate counting at different culture times showed that after BLa80 entered the stable period after 12h of culture, the flow counting results were higher than the plate counting results. Among them, the flow and plate counting results at 16h were 1.73×10 9 AFU / μL and 1.66×10 9 CFU / μL, the difference between the two was 4.21%. In summary, the similarity of the counting results of flow cytometry and plate method depends on the physiological state of bacteria, that is, the results of the two methods are closest when BLa80 is cultured for 16h.

[0130] In summary, the present invention provides a detection method based on flow cytometry and SYTO 9 / PI double staining technology, by detecting cells after SYTO 9 and PI double staining by flow cytometer, live bacteria (green fluorescence) and dead bacteria (red fluorescence) can be effectively distinguished, and high-throughput distinction between live and dead bacteria is achieved, which is used for rapid and accurate determination of live / dead bacteria counts of animal Bifidobacterium lactis subspecies, improves detection efficiency and accuracy, and provides strong technical support for quality control of probiotic fermentation products. Compared with the traditional plate count method, not only the number of live bacteria can be detected, but also the number of dead bacteria can be detected, with the characteristics of high efficiency and high accuracy.

[0131] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for rapid detection of live / dead bacteria in animal Bifidobacterium lactis fermentation broth, characterized in that: The method comprises: centrifuging the fermentation liquid and discarding the supernatant to obtain bacterial precipitation, diluting to obtain bacterial suspension, mixing with SYTO 9 / PI fluorescent dye, incubating, and analyzing by flow cytometry.

2. The method according to claim 1, characterized in that: The centrifugation and discarding of the supernatant specifically comprises: discarding the supernatant of the fermentation broth after the first centrifugation, resuspending the fermentation broth with the diluent, and collecting the precipitate by the second centrifugation; Preferably, the temperature of the first centrifugation and the second centrifugation is independently 0 to 8°C; Preferably, the rotation speeds of the first centrifugation and the second centrifugation are independently 5000-10000 r / min; Preferably, the time for the first centrifugation and the second centrifugation is independently 2 to 8 minutes.

3. The method according to claim 1 or 2, characterized in that: The dilution comprises: using a diluent to dilute the bacterial precipitate until the number of bacterial cells is 5×10 5 ~1×10 7 CFU / mL; Preferably, the diluent contains PBS buffer with a pH of 7-8.

4. The method according to any one of claims 1 to 3, characterized in that: The volume ratio of the bacterial suspension to the SYTO 9 / PI fluorescent dye is (500-1500):1; Preferably, the SYTO 9 / PI fluorescent dye comprises SYTO 9 dye and PI dye; Preferably, in the SYTO 9 / PI fluorescent dye, the volume ratio of SYTO 9 dye to PI dye is (3-5):1; Preferably, the preparation method of the SYTO 9 / PI fluorescent dye comprises: mixing SYTO 9 dye with a concentration of 3.34 mM and PI dye with a concentration of 20 mM in a volume ratio of (3-5):

1.

5. The method according to any one of claims 1 to 4, characterized in that: The incubation is light-proof incubation; Preferably, the incubation time is 20 to 30 minutes; Preferably, the incubation temperature is 20-30°C.

6. The method according to any one of claims 1 to 5, characterized in that: The excitation wavelength of the flow cytometer analysis was 488 nm, and the fluorescence channels were B525 and B585.

7. The method according to any one of claims 1 to 6, characterized in that: The setting parameters of the flow cytometer include: the total number of particles read is 20,000, the FSC channel gain is 1,000, the SSC channel gain is 1,000, the B525 channel gain is 30, and the B585 channel gain is 80.

8. The method according to any one of claims 1 to 7, characterized in that: The threshold values ​​of the flow cytometer were set as: FSC 50000, SSC 10000.

9. The method according to any one of claims 1 to 8, characterized in that: The compensation settings of the flow cytometer were: B525 6%, B585 64%.

10. The method according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: (1) Sample preparation: The fermented liquid of Bifidobacterium animalis subsp. lactis was centrifuged at 5000-10000 r / min for 2-8 min at 0-8°C, the supernatant was discarded, the liquid was resuspended in PBS buffer with a pH of 7-8, and the liquid was centrifuged at 5000-10000 r / min for 2-8 min at 0-8°C, the precipitate was collected, and the bacterial precipitate was diluted with PBS buffer with a pH of 7-8 until the bacterial count was 5×10 5 ~1×10 7 CFU / mL, and bacterial suspension was obtained; (2) Fluorescent staining: the bacterial suspension is mixed with SYTO 9 / PI fluorescent dye, incubated at 20-30° C. in the dark for 20-30 min, and SYTO 9 / PI fluorescent staining is performed, wherein the volume ratio of the bacterial suspension to the SYTO 9 / PI fluorescent dye is (500-1500):1, and the preparation method of the SYTO9 / PI fluorescent dye comprises mixing SYTO 9 dye with a concentration of 3.34 mM and PI dye with a concentration of 20 mM in a volume ratio of (3-5):1; (3) Flow cytometry: After incubation, the cells were analyzed by flow cytometry immediately with an excitation wavelength of 488 nm, fluorescence channels of B525 and B585, a total number of particles read of 20,000, FSC channel gain of 1,000, SSC channel gain of 1,000, B525 channel gain of 30, B585 channel gain of 80, thresholds set to FSC 50,000, SSC 10,000, and compensations set to B525 6% and B585 64%.

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