High-throughput screening method of intestinal lactic acid bacteria

Abstract

The invention discloses a high-throughput screening method of intestinal lactic acid bacteria. The method comprises the following steps: obtaining a to-be-detected sample with the intestinal lactic acid bacteria; enriching the sample to be detected; preparing a bacterial suspension according to the enriched sample, preparing an improved MRS culture medium, introducing the improved MRS culture medium into nine sterile plates, and marking the sterile plates; inoculating the prepared bacterial suspension into a corresponding sterile plate to obtain a screened intestinal flora; preparing a 0.08 g / L bromomethylphenol violet MRS broth culture medium, adding the medium into a porous plate, inoculating the screened intestinal flora into the porous plate, culturing for 24 hours, and determining the cultured flora of the porous plate by adopting a microplate reader; according to the method, the MRS broth culture medium with the optimal bromomethylphenol violet concentration is obtained through a control experiment, the lactic acid bacteria are screened out through color change of the culture medium and detection of the OD600 value in the culture medium, and the screening efficiency and accuracy of the lactic acid bacteria are improved.

Description

Technical Field

[0001] The invention belongs to the field of microbiology, and in particular relates to a high-throughput screening method for intestinal lactic acid bacteria. Background Art

[0002] As one of the probiotic strains that can be used for food, fermented lactobacillus can tolerate the low acid and high bile salt environment of the human intestinal tract and has strong adaptability in the gastrointestinal tract environment. Fermented lactobacillus has good beneficial effects in immunomodulation (colitis, irritable bowel syndrome, etc.), antagonism of pathogenic bacteria (Salmonella, Staphylococcus aureus, Candida, Streptococcus, etc.), anti-oxidation, cholesterol reduction, etc. As a probiotic, fermented lactobacillus has great research value in potential beneficial properties. Therefore, fast and efficient screening of excellent fermented lactobacillus becomes the problem to be solved first, and screening culture medium is the most critical in the screening process.

[0003] Traditional screening and separation steps include: collecting samples, diluting and coating (improving culture medium formula), picking single colonies one by one, purifying single colonies, PCR amplification (or designing lactobacillus specific primers), 16SrRNA sequencing, and large-scale blind screening. Traditional lactic acid bacteria screening methods are inefficient, time-consuming and costly. Therefore, there is an urgent need for a high-throughput screening method to quickly and effectively screen and isolate lactic acid bacteria strains with excellent properties. Summary of the invention

[0004] In order to solve the problems existing in the above prior art, the present invention proposes a high-throughput screening method for intestinal lactic acid bacteria, which comprises:

[0005] The first step is to obtain a sample to be tested containing intestinal lactic acid bacteria;

[0006] Step 2: Take 1 mL of the sample to be tested, put it into a test tube containing 9 mL of MRS broth medium, mix it evenly, and culture it at 37 degrees Celsius for 48 hours to obtain the enriched sample solution;

[0007] Step 3: Take 1 mL of the enriched sample solution and put it into a test tube containing 9 mL of sterile saline to dilute and mix to obtain 10 -1 of bacterial suspension; take 1mL of 10 -1 The bacterial suspension was placed in a test tube containing 9 mL of sterile saline and diluted and mixed to obtain 10 -2 The bacterial suspension was repeated to obtain 10 -3 and 10 -4 dilution;

[0008] Step 4: prepare a modified MRS medium containing 2 μg / mL vancomycin hydrochloride, introduce the modified MRS medium into 9 sterile plates, and mark the sterile plates;

[0009] Step 5: Use three 1 mL sterile pipettes to -2 , 10 -3 , 10 -4 0.1 mL of the diluted bacterial suspension was inhaled into each of the diluents and inoculated into the corresponding 9 sterile plates for 24 hours to obtain the screened intestinal flora;

[0010] Step 6: Prepare the optimal concentration of bromomethylphenol purple MRS broth medium and sterilize it by high pressure, wherein the optimal concentration of bromomethylphenol purple solution is 0.08 g / L, and add 100 μL of bromomethylphenol purple MRS broth medium into the multi-well plate in the clean bench;

[0011] Step 7: Use sterile toothpicks to inoculate the screened intestinal flora into multi-well plates and culture for 24 hours;

[0012] Step 8: Use an ELISA instrument to measure the bacterial colony on the multi-well plate after culture to obtain the OD600 value;

[0013] Step 9: Set the OD600 difference threshold, where the OD600 difference threshold is 0.2; calculate the difference between the measured OD600 value and the OD600 value of the standard bacteria, compare the calculated difference with the OD600 difference threshold, and identify the bacteria with color changes in the culture medium in the multi-well plate and OD600 values ​​reduced by more than 0.2 as lactic acid bacteria;

[0014] Step 10: Separate and purify the screened lactic acid bacteria, and sequence the separated and purified lactic acid bacteria.

[0015] Beneficial effects of the present invention:

[0016] The present invention additionally adds 2 μg / mL of vancomycin hydrochloride in a final concentration to the MRS culture medium formula, and cultures for only 24 hours, resulting in more lactic acid bacteria colonies in the shape of pinpoint bright beads, while non-lactic acid bacteria are inhibited and grow less; the present invention obtains an MRS broth culture medium containing an optimal bromomethylphenol purple concentration through a control experiment, and screens out lactic acid bacteria through the color change of the culture medium and the detection of the OD600 value in the culture medium, thereby improving the screening efficiency and accuracy of lactic acid bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the color change of the bacterial colony on the porous plate of the present invention;

[0018] Figure 2 The OD600 value trend diagram of bromomethylphenol violet with different final concentrations in the MRS broth medium of the present invention for 24 hours;

[0019] Figure 3It is a trend diagram of the absorbance difference between inoculated and uninoculated bacteria with different final concentrations of bromomethylphenol purple in the MRS broth medium of the present invention;

[0020] Figure 4 It is the overall flow chart of the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] A high-throughput screening method for intestinal lactic acid bacteria, such as Figure 4 As shown, the method includes:

[0023] The first step is to obtain a sample to be tested containing intestinal lactic acid bacteria;

[0024] Step 2: Take 1 mL of the sample to be tested, put it into a test tube containing 9 mL of MRS broth medium, mix it evenly, and culture it at 37 degrees Celsius for 48 hours to obtain the enriched sample solution;

[0025] Step 3: Take 1 mL of the enriched sample solution and put it into a test tube containing 9 mL of sterile saline to dilute and mix to obtain 10 -1 of bacterial suspension; take 1mL of 10 -1 The bacterial suspension was placed in a test tube containing 9 mL of sterile saline and diluted and mixed to obtain 10 -2 The bacterial suspension was repeated to obtain 10 -3 and 10 -4 dilution;

[0026] Step 4: prepare a modified MRS medium containing 2 μg / mL vancomycin hydrochloride, introduce the modified MRS medium into 9 sterile plates, and mark the sterile plates;

[0027] Step 5: Use three 1 mL sterile pipettes to -2 , 10 -3 , 10 -4 0.1 mL of the diluted bacterial suspension was inhaled into each of the diluents and inoculated into the corresponding 9 sterile plates for 48 hours to obtain the screened intestinal flora;

[0028] Step 6: Prepare the optimal concentration of bromomethylphenol purple MRS broth medium and sterilize it by high pressure, wherein the optimal concentration of bromomethylphenol purple solution is 0.08 g / L, and add 100 μL of bromomethylphenol purple MRS broth medium into the multi-well plate in the clean bench;

[0029] Step 7: Use sterile toothpicks to inoculate the screened intestinal flora into multi-well plates and culture for 24 hours;

[0030] Step 8: Use an ELISA instrument to measure the bacterial colony on the multi-well plate after culture to obtain the OD600 value;

[0031] Step 9: Set the OD600 difference threshold, where the OD600 difference threshold is 0.2; calculate the difference between the measured OD600 value and the OD600 value of the standard bacteria, compare the calculated difference with the OD600 difference threshold, and identify the bacteria with color changes in the culture medium in the multi-well plate and OD600 values ​​reduced by more than 0.2 as lactic acid bacteria;

[0032] Step 10: Separate and purify the screened lactic acid bacteria, and sequence the separated and purified lactic acid bacteria.

[0033] In this embodiment, obtaining a sample to be tested containing intestinal lactic acid bacteria includes: placing 30% glycerol in a stool sampling tube, placing 3 to 4 g of a stool sample in the stool sampling tube, and shaking the stool sampling tube to fully mix the stool sample with the glycerol; packaging the sample and storing it in a refrigerator at minus 80 degrees Celsius.

[0034] Specifically include:

[0035] 1. Choose a container: Use a clean, dry, sterile, covered container, such as a disposable plastic sterile stool sample box. Make sure there is no chemical residue in the container to avoid interfering with the sample test results.

[0036] 2. Collect samples: Use a sterile spoon in a fecal sampling tube (containing 30% glycerol as a protective agent) to scoop out the middle part of the fecal sample that is not in contact with the air and the ground. Scoop out a full spoonful of fecal sample, about the size of a broad bean (about 3-5g), and put it into the collection tube together with the spoon, and cover it tightly (shake the tube to fully mix the feces and glycerol). Avoid mixing impurities: Make sure that the fecal sample is not mixed with urine, menstrual blood, disinfectants, sewage, plants, soil and other impurities.

[0037] 3. Samples should be frozen and stored: After the samples are collected, they should be sent to the laboratory for packaging as soon as possible, generally within 2 hours. The samples should be frozen in a refrigerator at minus 80 degrees Celsius.

[0038] In this embodiment, the enrichment of the sample includes: vortexing the stool sample to mix evenly, taking 1 mL of the sample with a sterile pipette and adding it to a test tube containing 9 mL of sterile MRS broth medium, and shaking it fully on a vortex homogenizer to evenly disperse the sample, that is, 10 -1 dilution of the sample; culture at 37 degrees Celsius for 48 hours.

[0039] MRS broth contains:

[0040] Water: 1000 ml

[0041] Glucose: 20 g

[0042] Peptone: 10 g

[0043] Beef dip powder: 10g

[0044] Yeast extract powder: 5 g

[0045] Tween 80: 1 ml

[0046] Potassium hydrogen phosphate: 2 g

[0047] Diammonium hydrogen citrate: 2 g

[0048] Magnesium sulfate: 0.1 g

[0049] Manganese sulfate: 0.05 g

[0050] Sodium acetate: 2 g

[0051] In this embodiment, the preparation of the bacterial suspension includes: adding 9 mL of physiological saline to 4 test tubes, plugging and bandaging the test tubes, and sterilizing them under high pressure of 103 kPa and 121°C steam for 20 min to obtain sterile physiological saline; marking 10 in sequence on the 4 sterile test tubes containing 9 mL of physiological saline. -1 , 10 -2 , 10 -3 , 10 -4 Then use a sterile pipette to take 10 -1 Place 1 mL of bacterial suspension into a test tube containing 9 mL of sterile saline and dilute and mix to obtain 10 -2 Dilution, repeat this process to obtain 10 -3 ~10 -4 of dilution.

[0052] Modified MRS medium:

[0053] Water: 1000 ml

[0054] Glucose: 20 g

[0055] Peptone: 10 g

[0056] Beef dip powder: 10g

[0057] Yeast extract powder: 5 g

[0058] Tween 80: 1 ml

[0059] Potassium hydrogen phosphate: 2 g

[0060] Diammonium hydrogen citrate: 2 g

[0061] Magnesium sulfate: 0.1 g

[0062] Manganese sulfate: 0.05 g

[0063] Sodium acetate: 2 g

[0064] Agar: 15-20 g

[0065] Adjust pH to 5.2-5.4;

[0066] Specifically, the preparation of the culture medium is divided into 250 mL conical bottles, sealed with a sterile film and placed in a sterilizer for high temperature and high pressure sterilization, vancomycin hydrochloride is prepared into a 20 mg / mL solution, filtered and sterilized with a sterile filter head, and vancomycin hydrochloride is added to a sterile 50 degree Celsius MRS culture medium at a final concentration of 2 μg / mL, and the plates are shaken and poured; 9 sterile plates are taken and numbered 10 -2 , 10 -3 , 10 -4 Three sets each; Pour 9 plates of MRS medium modified with 2 μg / mL vancomycin hydrochloride according to the aseptic operation method, about 15 mL per dish; Place flat to evenly distribute the culture medium on the bottom of the dish and wait for solidification. In this embodiment, MRS medium with additional 2 μg / mL vancomycin hydrochloride is added for screening of intestinal flora. After 24 hours of cultivation, there are more single colonies of lactic acid bacteria, which are in the shape of pinpoint bright beads, and the miscellaneous bacteria of non-lactic acid bacteria are inhibited and grow less.

[0067] In this example, three 1 mL sterile pipettes or pipettes were used to draw 10 -2 , 10 -3 and 10 -4 Inoculate 0.1 mL of each diluted bacterial suspension into the corresponding 9 sterile plates, with 0.1 mL in each plate; spread the bacterial solution evenly on the plates with a sterile glass spreader as quickly as possible, and place them flat on the laboratory bench for 20 minutes; then invert and culture them in a 37°C incubator for 48 hours.

[0068] 0.08g / L bromomethylphenol purple MRS broth culture medium is:

[0069] Water: 1000 ml

[0070] Glucose: 20 g

[0071] Peptone: 10 g

[0072] Beef dip powder: 10g

[0073] Yeast extract powder: 5 g

[0074] Tween 80: 1 ml

[0075] Potassium hydrogen phosphate: 2 g

[0076] Diammonium hydrogen citrate: 2 g

[0077] Magnesium sulfate: 0.1 g

[0078] Manganese sulfate: 0.05 g

[0079] Sodium acetate: 2 g

[0080] Adjust pH to 7.2;

[0081] Prepare the culture medium and divide it into 250mL conical flasks, seal them with sterile film and place them in a sterilizer for high temperature and high pressure sterilization; prepare bromomethylphenol purple into a 16g / L solution, and add it to sterile MRS broth culture medium at a ratio of 5ml bromomethylphenol purple solution per liter of culture medium and shake well to obtain 0.08g / L bromomethylphenol purple MRS broth culture medium.

[0082] In this embodiment, the determination of the final concentration of bromomethylphenol purple includes: using an optically transparent and bacterioresistant sterile 96-well plate with a transparent cover to explore the final concentration of bromomethylphenol purple in MRS broth culture medium, and the specific data are shown in Table 1. Specifically comprising: adding MRS broth medium with pH 7.2 into a multi-well plate according to the final concentration of bromomethylphenol purple of 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 g / L, and each concentration was repeated three times; after 24 hours, the OD600 value of each well was measured; using MRS broth medium with bromomethylphenol purple at a final concentration of 0 g / L as a control and averaging the three repeated values ​​of each concentration after subtracting the background;

[0083] Table 1 OD600 values ​​of bromomethylphenol purple solution with different final concentrations

[0084]

[0085]

[0086] according to Figure 2The trend graph shows that the final concentration of bromomethylphenol purple in MRS broth medium is in the range of 0 to 0.1 g / L, and the OD600 value is proportional to its bromomethylphenol purple concentration, and tends to be flat after 0.11 g / L. Therefore, we choose the final concentration of bromomethylphenol purple in MRS broth medium to be 0 to 0.1 g / L.

[0087] In the experiment, 1 microliter of Lactobacillus rhamnosus (OD600=0.8) was inoculated into the wells containing 100 microliters of bromomethylphenol purple MRS broth medium in the 96-well plate, and then cultured at 37 degrees Celsius for 24 hours. The results showed that the color of bromomethylphenol purple in all wells containing Lactobacillus rhamnosus changed from purple to yellow. In these wells, the final concentration of bromomethylphenol purple ranged from 0.02g / L to 0.24g / L, and the corresponding OD600 absorbance values ​​showed different degrees of reduction, as shown in Table 2.

[0088] Table 2 OD600 difference between uninoculated and inoculated cultures in MRS broth medium with different concentrations of bromomethylphenol purple

[0089]

[0090]

[0091] The results are as follows Figure 3 As shown, the color of the bromomethylphenol purple MRS broth medium in the range of 0 to 0.06 g / L is lighter, and the color change cannot be clearly observed by the naked eye after turning yellow. According to the data in Table 2, when the bromomethylphenol purple concentration in the selected bromomethylphenol purple MRS broth medium is 0.08 g / L, the measured OD600 difference is 1.494, which is significantly increased relative to the OD600 difference of the bromomethylphenol purple solution of 0.07 g / L. Therefore, the color change of 0.08 g / L bromomethylphenol purple MRS broth medium is more obvious.

[0092] In this embodiment, the content of lactic acid bacteria is determined according to the difference in OD600 decrease. When the difference in OD600 decrease is 0.2, the percentage of lactic acid bacteria in all porous plates is 86.05%; when the difference in OD600 decrease is 0.3, the percentage of lactic acid bacteria in all porous plates is 85%; when the difference in OD600 decrease is 0.4, the percentage of lactic acid bacteria in all porous plates is 83.64%; when the difference in OD600 decrease is 0.5, the percentage of lactic acid bacteria in all porous plates is 83.18%. According to the experimental results and the corresponding data ratio, when the difference in OD600 decrease is 0.2, the proportion of lactic acid bacteria is high, and the color change in the culture solution when the OD600 absorbance decreases by more than 0.2 is obvious, so it is set that the OD600 absorbance decreases by more than 0.2 is acid-producing bacteria.

[0093] High-throughput screening of lactic acid bacteria includes: adding 1.6 grams of bromomethylphenol purple to 100 mL of 20% ethanol, shaking well, and adding it to the above-mentioned sterile MRS broth culture medium according to the final concentration of bromomethylphenol purple of 0.08 g / L. After shaking well, add it to 96-well plates, adding 100 microliters of liquid culture medium to each well. Use optically transparent and bacterioresistant sterile 96-well plates with transparent covers for culture and screening of strains. In the sterile 96-well plate, each well contains 100 microliters of MRS broth culture medium (containing 0.08g / L bromomethylphenol purple). According to the color, size, glossiness and transparency of the colony, pick a single colony and inoculate it in a 96-well plate with a sterile toothpick. Cultivate in a 37°C incubator for 24 hours, and the lactic acid bacteria grow and reproduce in the 96-well plate.

[0094] like Figure 1 As shown, by observing the growth of lactobacilli in a 96-well plate, such as the color change of the bacterial solution, etc. Normally, bromomethylphenol purple is yellow or yellow-green in an acidic environment, and blue or purple in a neutral or alkaline environment. Therefore, when lactic acid bacteria grow in a culture medium and produce lactic acid, the pH of the liquid culture medium will decrease, causing bromomethylphenol purple to change from blue or purple to yellow or yellow-green.

[0095] The absorbance value of OD600 in a 96-well plate was measured by an ELISA instrument. When measured in a liquid culture medium, the color change of bromomethylphenol purple will cause the OD600 value to decrease. The organic acids such as lactic acid produced during the growth and metabolism of lactic acid bacteria will affect the transparency of the culture medium, and the decrease in pH value will cause the color of the bromomethylphenol purple solution to become lighter, thereby reducing the OD600 value. Therefore, the combination of color change and a decrease in OD600 value of more than 0.2 can directly determine the presence of acid-producing bacteria.

[0096] In this embodiment, the screening colonies are separated and purified by using a sterile inoculation loop to dip into a single-well liquid culture medium that has changed color or whose OD600 value has decreased by more than 0.2, and the microorganisms that grow and form single colonies on an MRS solid culture medium are separated by a partitioning and streaking method.

[0097] The isolated and purified single colony was inoculated into MRS broth medium for culture. The cultured bacterial suspension was amplified by PCR and sent to a sequencing company for sequencing.

[0098] Specifically, 1 mL of bacterial solution was centrifuged at 10,000 r / min for 1 min, the supernatant was discarded to obtain the bacteria, and 500 μL of sterile water was added to resuspend the bacteria as a bacterial solution template.

[0099] Take 2 μl of the resuspended bacterial suspension as a template for 16S rDNA PCR as follows:

[0100] Take 2 μl of resuspended bacterial suspension as template

[0101] System 50μL

[0102] Taq mix 25 μl

[0103] ddH2O 19μl

[0104] 1492R primer 2 μl

[0105] 27F primer 2 μl

[0106] Bacterial suspension template 2μl

[0107] PCR conditions: 94°C for 5 min; 94°C for 30 s; 55°C for 30 s; 72°C for 2 min; 72°C for 10 min; 2-4 steps for 30 cycles.

[0108] Sequencing and analysis: After the PCR product is confirmed by nucleic acid electrophoresis analysis, it is sent for sequencing.

[0109] Experimental verification:

[0110] According to the above test results, the OD600 difference threshold was set to 0.2, the final concentration of bromomethylphenol purple was configured to 0.08g / L, and 9 healthy college students were selected and separated according to the methods of steps 1 to 5 above. Ten pinpoint water droplet-shaped colonies were randomly selected from each sample, for a total of 90 single colonies, which were inoculated into a 96-well plate according to step 6, and the OD600 absorbance value was measured with a microplate reader. The 86 strains with an OD600 absorbance value lower than 0.2 were separated, purified and identified according to steps 7 to 10.

[0111] The difference between the bacterial species identification results and the OD600 decrease is shown in the following table.

[0112]

[0113]

[0114] In this example, 68 strains were identified as lactic acid bacteria. This method significantly improves the efficiency of bacterial separation and provides an effective way for bacterial identification and research.

[0115] The above embodiments further illustrate the purpose, technical solutions and advantages of the present invention in detail. It should be understood that the above embodiments are only preferred implementation modes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made to the present invention within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A high-throughput screening method for intestinal lactic acid bacteria, characterized in that: include: The first step is to obtain a sample to be tested containing intestinal lactic acid bacteria; Step 2: Take 1 mL of the sample to be tested, put it into a test tube containing 9 mL of MRS broth medium, mix it evenly, and culture it at 37 degrees Celsius for 48 hours to obtain the enriched sample solution; Step 3: Take 1 mL of the enriched sample solution and put it into a test tube containing 9 mL of sterile saline to dilute and mix to obtain 10 -1 of bacterial suspension; take 1mL of 10 -1 The bacterial suspension was placed in a test tube containing 9 mL of sterile saline and diluted and mixed to obtain 10 -2 The bacterial suspension was repeated to obtain 10 -3 and 10 -4 dilution; Step 4: prepare a modified MRS medium containing 2 μg / mL vancomycin hydrochloride, introduce the modified MRS medium into 9 sterile plates, and mark the sterile plates; Step 5: Use three 1 mL sterile pipettes to -2 , 10 -3 , 10 -4 0.1 mL of the diluted bacterial suspension was inhaled from each dilution and inoculated into the corresponding 9 sterile plates for 48 hours. Each dilution corresponded to 3 sterile plates to obtain the screened intestinal flora. Step 6: Prepare the optimal concentration of bromomethylphenol purple MRS broth medium and sterilize it by high pressure, wherein the optimal concentration of bromomethylphenol purple solution is 0.08 g / L; add 100 μL of bromomethylphenol purple MRS broth medium into the multi-well plate in the clean bench; Step 7: Use sterile toothpicks to inoculate the screened intestinal flora into multi-well plates and culture for 24 hours; Step 8: Use an ELISA instrument to measure the bacterial colony on the multi-well plate after culture to obtain the OD600 value; Step 9: Set the OD600 difference threshold, where the OD600 difference threshold is 0.2; The difference between the measured OD600 value and the OD600 value of the blank culture medium is calculated, and the calculated difference is compared with the OD600 difference threshold value, and the bacterial colonies with color changes in the culture medium in the multi-well plate and OD600 values ​​reduced by more than 0.2 are identified as lactic acid bacteria; Step 10: Separate and purify the screened lactic acid bacteria, and sequence the separated and purified lactic acid bacteria.

2. The high-throughput screening method for intestinal lactic acid bacteria according to claim 1, characterized in that: The method of obtaining a test sample containing intestinal lactic acid bacteria includes: placing 30% glycerol in a stool sampling tube, placing 3 to 4 g of a stool sample in the stool sampling tube, and shaking the stool sampling tube to fully mix the stool sample with the glycerol; packaging the sample and storing it in a refrigerator at minus 80 degrees Celsius.

3. The high-throughput screening method for intestinal lactic acid bacteria according to claim 1, characterized in that: MRS broth medium includes: Water: 1000 ml Glucose: 20 g Peptone: 10 g Beef dip powder: 10g Yeast extract powder: 5 g Tween 80: 1 ml Potassium hydrogen phosphate: 2 g Diammonium hydrogen citrate: 2 g Magnesium sulfate: 0.1 g Manganese sulfate: 0.05 g Sodium acetate: 2 g.

4. The high-throughput screening method for intestinal lactic acid bacteria according to claim 1, characterized in that: The method of obtaining sterile saline solution includes: adding 9 mL of saline solution into 4 test tubes, plugging and bandaging the mouths of the test tubes, and sterilizing them under high pressure of 103 kPa and 121°C steam for 20 minutes to obtain sterile saline solution.

5. The high-throughput screening method for intestinal lactic acid bacteria according to claim 1, characterized in that: Modified MRS medium: Water: 1000 ml Glucose: 20 g Peptone: 10 g Beef dip powder: 10g Yeast extract powder: 5 g Tween 80: 1 ml Potassium hydrogen phosphate: 2 g Diammonium hydrogen citrate: 2 g Magnesium sulfate: 0.1 g Manganese sulfate: 0.05 g Sodium acetate: 2 g Agar: 15-20 g Adjust pH to 5.2-5.4; The prepared culture medium was divided into 250 mL conical flasks, sealed with sterile membranes and placed in a sterilizer for high temperature and high pressure sterilization. Vancomycin hydrochloride was prepared into a 20 mg / mL solution, filtered and sterilized with a sterile filter head, and then vancomycin hydrochloride was added to a sterile MRS medium at 50 degrees Celsius at a final concentration of 2 μg / mL and shaken to obtain a modified MRS medium.

6. The high-throughput screening method for intestinal lactic acid bacteria according to claim 1, characterized in that: 0.08g / L bromomethylphenol purple MRS broth culture medium is: Water: 1000 ml Glucose: 20 g Peptone: 10 g Beef dip powder: 10g Yeast extract powder: 5 g Tween 80: 1 ml Potassium hydrogen phosphate: 2 g Diammonium hydrogen citrate: 2 g Magnesium sulfate: 0.1 g Manganese sulfate: 0.05 g Sodium acetate: 2 g Adjust pH to 7.2; Prepare the culture medium and divide it into 250mL conical flasks, seal them with sterile film and place them in a sterilizer for high temperature and high pressure sterilization; prepare bromomethylphenol purple into a 16g / L solution, and add it to sterile MRS broth culture medium at a ratio of 5ml bromomethylphenol purple solution per liter of culture medium and shake well to obtain 0.08g / L bromomethylphenol purple MRS broth culture medium.

7. A high-throughput screening method for intestinal lactic acid bacteria according to claim 6, characterized in that: The method for obtaining a 0.08 g / L bromomethylphenol purple solution comprises: adding MRS broth medium with a pH of 7.2 to a multiwell plate at a final concentration of bromomethylphenol purple of 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, and 0.24 g / L, respectively, and repeating each concentration three times; after 24 hours, measuring the OD600 value of each well; using MRS broth medium with a final concentration of 0 g / L bromomethylphenol purple as a control and subtracting the background, The three repeated values ​​of the concentration were averaged to obtain the bromomethylphenol purple concentration after preliminary screening; different control experiments were divided according to the bromomethylphenol purple concentrations obtained by preliminary screening, and 1 microliter of Lactobacillus rhamnosus was inoculated into the wells containing 100 microliters of bromomethylphenol purple MRS broth culture medium in the control experiment, wherein Lactobacillus rhamnosus was the standard bacteria, and after culturing at 37 degrees Celsius for 24 hours, the OD600 value of each control experiment was measured, and according to the measurement results, the concentration of the bromomethylphenol purple solution of the control experiment with obvious color change of the culture medium and a higher decrease in OD600 value was selected as the optimal concentration; that is, 0.08 g / L bromomethylphenol purple MRS broth culture medium was used to screen lactic acid bacteria, and the bacterial population with an OD600 value decrease of more than 0.2 was selected as lactic acid bacteria.

8. The high-throughput screening method for intestinal lactic acid bacteria according to claim 1, characterized in that: The multi-well plate is a 96-well plate.

9. The high-throughput screening method for intestinal lactic acid bacteria according to claim 1, characterized in that: The separation and purification of lactic acid bacteria includes: using a sterile inoculation loop to dip into a single-well liquid culture medium that has changed color or whose OD600 value has decreased by more than 0.2, and growing and forming a single colony of microorganisms on an MRS solid culture medium by a zoning and streak separation method; inoculating the separated and purified single colony into an MRS broth culture medium for cultivation; taking 1 mL of bacterial solution and centrifuging at 10,000 r / min for 1 minute, discarding the supernatant to obtain the bacterial body, adding 500 μL of sterile water to resuspend it, and using it as a bacterial solution template; performing 16S rDNA PCR system amplification according to the bacterial solution template, and performing nucleic acid electrophoresis analysis on the amplified product.

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