Method for screening co-metabolism degrading bacteria through optical tweezers single cells

Abstract

The invention relates to a microorganism screening and culturing method, in particular to a method for screening co-metabolism degrading bacteria through optical tweezers single cells. Comprising the following steps: (1) inoculating a to-be-screened strain sample to a culture medium containing a to-be-degraded substance, and culturing; (2) carrying out single-cell capture on the strain cultured in the step (1) by adopting optical tweezers, and then collecting the captured single strain through microfluidics; and (3) respectively carrying out amplification culture on the plurality of single strains collected in the step (2). According to the method disclosed by the invention, the screening efficiency of the co-metabolism degrading bacteria is remarkably improved, and meanwhile, the screening precision and real-time performance of the co-metabolism degrading bacteria are also improved.

Description

Technical Field

[0001] The invention relates to a microorganism screening and culturing method, and in particular to an optical tweezers single cell screening method for co-metabolism degradation bacteria. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Cometabolic degraders refer to a special type of microorganisms that do not directly use the pollutants themselves as their energy or carbon source during the process of degrading pollutants, but rely on the auxiliary role of certain major metabolic substrates. This mechanism enables cometabolic degraders to treat many difficult-to-degrade organic pollutants that traditional microorganisms cannot directly degrade. These microorganisms play an important ecological role in the natural environment. They can not only improve the removal efficiency of pollutants, but also have strong environmental adaptability. Therefore, in pollution remediation technology, cometabolic degraders, as a highly selective and efficient tool, show great application potential.

[0004] Traditional screening methods rely on manual operations and long culture processes, and are usually only used for screening specific types of cells. They have limitations such as low throughput, large amounts of time and manpower, etc. In addition, traditional methods still produce mixed bacteria even after repeated streaking separation. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a method for single-cell screening of cometabolic degrading bacteria using optical tweezers. The method of the present invention significantly improves the screening efficiency of cometabolic degrading bacteria, while also improving the screening accuracy and real-time performance of cometabolic degrading bacteria.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A method for screening co-metabolism degradation bacteria by single-cell optical tweezers comprises the following steps:

[0008] (1) inoculating a sample of the strain to be screened into a culture medium containing a substance to be degraded for culturing;

[0009] (2) using optical tweezers to capture single cells of the strain cultured in step (1), and then collecting the captured single strain through microfluidics;

[0010] (3) Expand and culture the several individual strains collected in step (2) respectively.

[0011] Single cell sorting technology can act directly on individual cells, avoiding the interference of group effects, making the evaluation of cell traits more accurate and reliable. The outstanding advantage of single cell sorting is that it can directly "measure" individual cells, quickly and accurately identify and separate cells with specific traits, greatly shorten the screening cycle, and improve screening efficiency. In addition, single cell sorting technology can act directly on individual cells, avoiding the interference of group effects, making the evaluation of cell traits more accurate and reliable. The techniques for single cell sorting include micromanipulation technology, fluorescence activated cell sorting, microfluidic sorting, optical tweezers sorting, cell sorting based on laser-induced forward transfer technology, etc. However, the present invention has found that it is difficult to achieve the screening of co-metabolism degradation bacteria using a single single cell sorting technology.

[0012] For example, optical tweezers can not only accurately capture the target strain, but also monitor the activity and morphological changes of cells in real time in combination with microscopic imaging; this enables optical tweezers to directly separate the target degrading bacteria in a mixed microbial population and retain their integrity and activity. However, the traditional optical tweezers method of sorting single cells is to transport the target single cells to the collection area, and the non-target single cells enter the waste liquid area; it can only separate one type of single cell at a time, and cannot separate multiple single cells at the same time; and to obtain and screen strains with specific functions, it is necessary to perform functional screening on multiple strains at the same time, and there are multiple strains with this function. Therefore, the traditional optical tweezers method of sorting single cells requires multiple optical tweezers sorting to screen multiple strains, which cannot improve the screening efficiency.

[0013] Microfluidic sorting has the advantages of flexible operation, integration, and fast speed. However, microorganisms in nature usually have different shapes and contain many microparticle impurities of different sizes. During sorting, pipeline blockage and other problems are prone to occur, which will also affect the screening efficiency.

[0014] Therefore, the present invention uses the advantage of optical tweezers to accurately capture single cells to capture single strains, and moves them to the channel of microfluidics, and quickly collects the captured single strains through the advantage of microfluidics, which not only solves the defect that optical tweezers cannot screen multiple strains at the same time, but also avoids the blockage problem caused by excessive strains or the presence of microparticle impurities, so that the efficiency of separating several single strains from the strain sample to be screened is significantly improved, thereby significantly improving the screening efficiency of co-metabolism degradation bacteria. The single-cell separation technology combining microfluidics and optical tweezers is a complementary advantage of the two. Compared with the use of microfluidics or optical tweezers alone, this method not only improves the efficiency and accuracy of the operation, but also increases the scope of application and experimental flexibility, providing a more powerful tool for single-cell research.

[0015] The beneficial effects of the present invention are:

[0016] The present invention realizes the "screening first and then separation" of degrading bacteria by combining the traditional culture method with the optical tweezers single-cell separation method. The traditional screening method often takes more than half a year to obtain pure bacteria, and is only suitable for the screening of sole carbon source degrading bacteria. Compared with the traditional screening method, the screening method only takes one week, is more efficient and is suitable for screening sole carbon source or co-metabolism degrading bacteria, which improves the accuracy, timeliness and efficiency of screening degrading bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 A schematic diagram of a microbial screening method according to an embodiment of the present invention;

[0019] Figure 2 This is a diagram of the optical tweezers-microfluidic single-cell separation process of Example 1 of the present invention. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0022] In view of the low screening efficiency of cometabolic degradation bacteria in the prior art, the present invention proposes a method for screening cometabolic degradation bacteria by single cell using optical tweezers.

[0023] A typical embodiment of the present invention provides a method for screening co-metabolism degradation bacteria by single-cell optical tweezers, comprising the following steps:

[0024] (1) inoculating a sample of the strain to be screened into a culture medium containing a substance to be degraded for culturing;

[0025] (2) using optical tweezers to capture single cells of the strain cultured in step (1), and then collecting the captured single strain through microfluidics;

[0026] (3) Expand and culture the several individual strains collected in step (2) respectively.

[0027] The present invention firstly cultures the strain sample to be screened with a culture medium containing the substance to be degraded, thereby screening out strains capable of degrading the target substance, and then separates several individual strains capable of degrading the target substance by means of optical tweezers-microfluidics. Through the collaboration of optical tweezers technology and microfluidics technology, the co-metabolic degradation strains capable of degrading the target substance can be screened more accurately, more quickly and in real time.

[0028] In some embodiments, the culture in step (1) includes enrichment culture and subculture. This culture method is more conducive to the screening of co-metabolism degradation strains.

[0029] In some embodiments, the culture medium in step (1) comprises: NH4Cl 0.27-0.33 g / L, KH2PO4 0.18-0.22 g / L, Na2HPO4 0.12-0.14 g / L, NaCl 0.9-1.1 g / L, MgCl2·6H2O 0.45-0.55 g / L, CaCl2·2H2O 0.013-0.017 g / L, and the rest is water. The pH value is 6.9-7.1.

[0030] In some embodiments, the co-metabolism carbon source of the target degrading bacteria is glucose, methanol, ethanol, lactic acid, acetate or propionate. Specifically, it can be 5-20 mM glucose, 2-10 mM methanol, 1-5 mM ethanol, 1-10 mM lactic acid, 1-10 mM acetate, 0.5-5 mM propionate.

[0031] In some embodiments, the process of step (1) is: adding aerobic sludge to an enrichment culture medium containing pollutants, culturing at 20-37°C and 140-210 r / min for 7-10 days, transferring to fresh culture medium several times to obtain an enriched bacterial liquid.

[0032] Specifically, the transfer inoculation amount is 5-10wt%, and the transfer times are 3-4 times.

[0033] In some embodiments, the strains cultured in step (1) are separated by streaking using optical tweezers, and then single cells are captured using optical tweezers, and the captured single strains are then collected by microfluidics. The streaking separation is beneficial for pre-screening and removing strains that obviously have the target function, further improving the screening efficiency.

[0034] Specifically, before streaking, the bacterial solution cultured in step (1) is diluted, and then spread on LB solid medium for culture, and then streaking is performed. More specifically, the culture temperature is 20-37° C. More specifically, in the LB solid medium, yeast powder 9-11 g / L, peptone 4.5-5.5 g / L, sodium chloride 9.0-11.0 g / L, agar 10-15.0 g / L, and the rest is water; the pH is 6.0-8.0.

[0035] In some embodiments, in step (2), the cultured bacterial solution is added to the liquid inlet of the microfluidic chip, observed under a microscope and single cells are captured using optical tweezers, and moved to the main channel of the microfluidic chip and then collected by microfluidics.

[0036] Specifically, the microscope is a bright field microscope or a fluorescence microscope.

[0037] Specifically, the microscope was used for observation using a 20x or 50x objective lens.

[0038] Specifically, the optical tweezers use 1060-1070nm laser 1900-2100mA.

[0039] Specifically, the liquid flowing in the microfluidics is PBS buffer.

[0040] Specifically, the liquid flowing in the microfluidics flows at a speed of 5-10 μL / min.

[0041] Specifically, Tween-20 is added to the bacterial solution after culture to prevent cells from adhering to the chip. More specifically, the addition amount is 0.18-0.22wt%

[0042] In some embodiments, LB medium is used for expansion culture.

[0043] In some embodiments, the time for expansion culture is 7-10 days.

[0044] In some embodiments, the step of verifying the degradation effect of the strain is also included. The degradation effect of the screened strain can be verified. Specifically, a culture medium containing the substance to be degraded is used for cultivation. After the bacterial liquid is centrifuged after cultivation, the supernatant is taken and the supernatant is tested. For example, if the substance to be degraded is tetrachloroundecanoic acid, the F of the supernatant is measured using a HQ30D portable multimeter (HACH) connected to an ion selective electrode (ISE, HACH). - Take measurements.

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0046] Example 1

[0047] Screening of microorganisms that degrade tetrachloroundecanoic acid

[0048] (1) Enrichment, subculture and agar dilution:

[0049] 6700 mg / L activated sludge (10%, v / v) was collected from an aerobic pool of a sewage plant in Qingdao and inoculated into a 150 mL serum bottle containing 90 mL of high-pressure sterilized (121°C, 20 min) inorganic salt culture medium (NH4Cl 0.3 g / L, KH2PO4 0.2 g / L, Na2HPO4 0.13 g / L, NaCl 1 g / L, MgCl2·6H2O 0.5 g / L, CaCl2·2H2O 0.015 g / L, and the rest was water; pH value was 7.0), which contained 100 μg / L vitamin B 12 . About 5 mM methanol was added to the culture as an additional carbon source and re-added every two weeks. About 50 μM of the selected chlorofluorinated organic (tetrachloroundecanoic acid) was spiked into the culture. The culture pH was buffered at 7.4 ± 0.1. The culture conditions were 30 ° C, 180 r / min, and shaker culture for 7 days. After the defluorination reached the maximum level of about 80%, the last subculture was further transferred (5%, v / v) to the same culture medium to obtain subsequent subcultures. Dilute 10 with sterile water -2 , 10 -3 , 10 -4 , spread on LB solid culture medium (yeast powder 10g / L, peptone 5.0g / L, sodium chloride 10.0g / L, agar 10-15.0g / L, the rest is water; pH is 7.0), culture at 30℃, pick single colonies with different colors and shapes, and separate them by repeated streaking.

[0050] (2) Optical tweezers-microfluidic single cell separation:

[0051] Take 1 mL of bacterial solution and add it to a 1.5 mL sterile centrifuge tube. Centrifuge at 4000 rpm for 10 min at room temperature, and then wash the bacterial solution three times with sterile water. Use PBS buffer to adjust the sample concentration to 5.0 × 10 8cells / mL and add a total of 0.2wt% Tween-20 to avoid cell adhesion in the chip. Take 20μL of sterile water and drop it on the liquid inlet of the microfluidic chip, take 20μL of sample and drop it on the sample outlet of the microfluidic chip, then put the microfluidic chip into the vacuum injection device, and the vacuum degree reaches -95kPa, so that the sample enters the sample pool in the chip to complete the injection. Use a syringe to draw 1mL of buffer and fix the syringe on the instrument injection pump. After expelling the bubbles inside the syringe, connect the hose and insert the other end of the hose into the liquid inlet of the chip. Keep the buffer flow rate at 9μL / min, run for 30 minutes to flush the microfluidic chip channel, then insert the capillary into the sample outlet of the chip and run for another 30 minutes to flush the capillary. Observe the sample under a 50x objective lens, set the injection pump flow rate to 9μL / min, and set the drag speed to 0.05mm / s. Figure 2 As shown, the single cells that enter the chip will be stored in the cell pool ( Figure 2 A); A single cell was captured at X2 using optical tweezers (1064nm laser 2000mA) Figure 2 B); drag a single cell through the channel ( Figure 2 C); finally release the optical tweezers when reaching X1 ( Figure 2 D), the single cells will flow out with the buffer and be collected in a 96-well plate. The sorted single cells are cultured in a single well, and 200 μL LB medium is added to each well.

[0052] (3) Verification of strain degradation effect

[0053] The bacterial suspension obtained by separation and culture was centrifuged at 8000g for 3 min at room temperature and then washed with 0.9% physiological saline for 3 times. The bacterial suspension (10%, v / v) was inoculated into 100 ml of inorganic salt liquid culture medium containing 50 μM tetrachloroundecanoic acid. After 30 days of culture, 2 mL of bacterial suspension was collected and then centrifuged at 16000g for 35 min at 4°C. The supernatant was collected and then measured using an HQ30D portable multimeter (HACH) connected to an ion selective electrode (ISE, HACH). - A total of 48 strains that have a degrading effect on tetrachloroundecanoic acid were screened out. These strains were mainly divided into four genera through high-throughput sequencing: Acinetobacter sp., Lysinibacillus sp., Comamonas sp. and Aquamicrobium sp., among which Aquamicrobium sp. had the best defluorination effect, with a defluorination rate of 50% in 30 days.

[0054] Example 2

[0055] Screening of microorganisms that degrade tetrachloroundecanoic acid

[0056] (1) Enrichment, subculture and agar dilution:

[0057] 6800 mg / L activated sludge (10%, v / v) was collected from an aerobic pool of a sewage plant in Qingdao and inoculated into a 150 mL serum bottle containing 90 mL of high-pressure sterilized (121°C, 20 min) inorganic salt medium containing 100 μg / L vitamin B 12 . About 5 mM methanol was added to the culture as an electron donor and re-added every two weeks. About 50 μM of the selected chlorofluorinated organic (tetrachloroundecanoic acid) was spiked into the culture. The culture pH was buffered at 7.4 ± 0.1. The culture conditions were 30 ° C, 180 r / min, and shaker culture for 7 days. After the defluorination reached the maximum level of about 80%, the last subculture was further transferred (5%, v / v) to the same culture medium to obtain subsequent subcultures. Dilute 10 with sterile water -2 , 10 -3 , 10 -4 , spread onto LB solid culture medium, culture at 30°C, pick out single colonies with different colors and shapes, and separate them by repeated streaking.

[0058] (2) Optical tweezers-microfluidic single cell separation:

[0059] The bacterial suspension obtained by separation and culture was centrifuged at 8000g for 3 minutes at room temperature, and then washed with physiological saline for 3 times. The bacterial suspension (10%) was inoculated v / v into 100ml inorganic salt liquid culture medium containing 50μM tetrachloroundecanoic acid. After 30 days of culture, 1mL of bacterial suspension was added to a 1.5mL sterile centrifuge tube, centrifuged at 8000rpm for 5 minutes at room temperature, and then washed with sterile water for 3 times. The sample concentration was adjusted to 5.0×10 6cells / mL and add a total of 0.2wt% Tween-20 to avoid cell adhesion in the chip. Take 10μL of sterile water and drop it on the inlet of the microfluidic chip, take 10μL of sample and drop it on the outlet of the microfluidic chip, then put the microfluidic chip into the vacuum injection device, the vacuum degree reaches -95kPa, so that the sample enters the sample pool in the chip to complete the injection. Use a syringe to draw 1mL of buffer and fix the syringe on the instrument injection pump. After the bubbles inside the syringe are discharged, connect the hose and insert the other end of the hose into the inlet of the chip. Keep the buffer flow rate at 9μL / min, run for 10min to flush the microfluidic chip channel, then insert the capillary into the outlet of the chip and run for another 10min to flush the capillary. Observe the sample under a 50x objective lens, set the injection pump flow rate to 9μL / min, and set the drag speed to 0.01mm / s. The single cell that enters the chip will be stored in the cell pool; the single cell is captured by optical tweezers (1064nm laser 2000mA) at X2; the single cell is dragged through the channel; finally, the optical tweezers are released at X1, and the single cell will flow out with the buffer and be collected in a 96-well plate. The sorted single cells are cultured in a single well, and 50μL LB culture medium is added to each well.

[0060] (3) Verification of strain degradation effect

[0061] The bacterial suspension obtained by separation and culture was centrifuged at 8000g for 3 min at room temperature and then washed with 0.9% physiological saline for 3 times. The bacterial suspension (10%, v / v) was inoculated into 100 ml of inorganic salt liquid culture medium containing 50 μM tetrachloroundecanoic acid. After 30 days of culture, 2 mL of bacterial suspension was collected and then centrifuged at 16000g for 35 min at 4°C. The supernatant was collected and then measured using an HQ30D portable multimeter (HACH) connected to an ion selective electrode (ISE, HACH). - .

[0062] Example 3

[0063] Screening of microorganisms capable of degrading 5,5,5-trifluorovaleric acid

[0064] (1) Enrichment, subculture and agar dilution:

[0065] 6700 mg / L activated sludge (10%, v / v) was collected from an aerobic pool of a sewage plant in Qingdao and inoculated into a 150 mL serum bottle containing 90 mL of high-pressure sterilized (121°C, 20 min) inorganic salt culture medium (NH4Cl 0.3 g / L, KH2PO4 0.2 g / L, Na2HPO4 0.13 g / L, NaCl 1 g / L, MgCl2·6H2O 0.5 g / L, CaCl2·2H2O 0.015 g / L, and the rest was water; pH value was 7.0), which contained 100 μg / L vitamin B 12 . About 5 mM methanol was added to the culture as an additional carbon source and re-added every two weeks. About 50 μM of the selected 5,5,5-trifluoropentanoic acid was spiked into the culture. The culture pH was buffered at 7.4 ± 0.1. The culture conditions were 30 ° C, 180 r / min, and shaker culture for 7 days. After defluorination reached a maximum level of about 80%, the last subculture was further transferred (5%, v / v) to the same culture medium to obtain subsequent subcultures. Dilute 10 with sterile water -2 , 10 -3 , 10 -4 , spread on LB solid culture medium (yeast powder 10g / L, peptone 5.0g / L, sodium chloride 10.0g / L, agar 10-15.0g / L, the rest is water; pH is 7.0), culture at 30℃, pick single colonies with different colors and shapes, and separate them by repeated streaking.

[0066] (2) Optical tweezers-microfluidic single cell separation:

[0067] Take 1 mL of bacterial solution and add it to a 1.5 mL sterile centrifuge tube. Centrifuge at 4000 rpm for 10 min at room temperature, and then wash the bacterial solution three times with sterile water. Use PBS buffer to adjust the sample concentration to 5.0 × 10 8 cells / mL and add a total of 0.2wt% Tween-20 to avoid cell adhesion in the chip. Take 20μL of sterile water and drop it on the liquid inlet of the microfluidic chip, take 20μL of sample and drop it on the sample outlet of the microfluidic chip, then put the microfluidic chip into the vacuum injection device, and the vacuum degree reaches -95kPa, so that the sample enters the sample pool in the chip to complete the injection. Use a syringe to draw 1mL of buffer and fix the syringe on the instrument injection pump. After expelling the bubbles inside the syringe, connect the hose and insert the other end of the hose into the liquid inlet of the chip. Keep the buffer flow rate at 9μL / min, run for 30 minutes to flush the microfluidic chip channel, then insert the capillary into the sample outlet of the chip and run for another 30 minutes to flush the capillary. Observe the sample under a 50x objective lens, set the injection pump flow rate to 9μL / min, and set the drag speed to 0.05mm / s. Figure 2 As shown, the single cells that enter the chip will be stored in the cell pool ( Figure 2 A); A single cell was captured at X2 using optical tweezers (1064nm laser 2000mA) Figure 2 B); drag a single cell through the channel ( Figure 2 C); finally release the optical tweezers when reaching X1 ( Figure 2 D), the single cells will flow out with the buffer and be collected in a 96-well plate. The sorted single cells are cultured in a single well, and 200 μL LB medium is added to each well.

[0068] (3) Verification of strain degradation effect

[0069] The bacterial suspension obtained by separation and culture was centrifuged at 8000g for 3 min at room temperature and then washed with 0.9% saline for 3 times. The bacterial suspension (10%, v / v) was inoculated into 100 ml of inorganic salt liquid culture medium containing 50 μM 5,5,5-trifluorovaleric acid. After 48 days of culture, 2 mL of bacterial suspension was collected and then centrifuged at 16000g for 35 min at 4°C. The supernatant was collected and then measured using an HQ30D portable multimeter (HACH) connected to an ion selective electrode (ISE, HACH). - Pseudomonas aeruginosa was screened out, and the defluorination rate could reach 33% in 48 days.

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for screening co-metabolism degradation bacteria by single-cell optical tweezers, characterized in that: The steps include: (1) inoculating a sample of the strain to be screened into a culture medium containing a substance to be degraded for culturing; (2) using optical tweezers to capture single cells of the strain cultured in step (1), and then collecting the captured single strain through microfluidics; (3) Expand and culture the several individual strains collected in step (2) respectively.

2. The method according to claim 1, characterized in that: The cultivation in step (1) includes enrichment cultivation and subculture.

3. The method according to claim 1, characterized in that: The process of step (1) is: adding aerobic sludge to the enrichment culture medium containing pollutants, culturing at 20-37°C and 140-210r / min for 7-10 days, transferring to fresh culture medium several times, and obtaining enriched bacterial liquid.

4. The method according to claim 1, characterized in that: The strains cultured in step (1) are separated by streaking using optical tweezers, and then single cells are captured using optical tweezers. The captured single strains are then collected by microfluidics.

5. The method according to claim 4, characterized in that: Before streaking, the bacterial solution cultured in step (1) is diluted and then spread onto LB solid culture medium for culture, followed by streaking.

6. The method according to claim 1, characterized in that: In step (2), the cultured bacterial solution is added to the liquid inlet of the microfluidic chip, observed using a microscope and single cells are captured using optical tweezers, and moved to the main channel of the microfluidic chip, and then collected by microfluidics.

7. The method according to claim 6, characterized in that: The microscope is a bright field microscope or a fluorescence microscope; Or, optical tweezers use 1060-1070nm laser 1900-2100mA; Or, the liquid flowing in the microfluidics flows at a rate of 5-10 μL / min; Alternatively, Tween-20 can be added to the bacterial solution after culture.

8. The method according to claim 1, characterized in that: LB medium was used for expansion culture.

9. The method according to claim 1, characterized in that: The time for expansion culture is 7-10 days.

10. The method according to claim 1, characterized in that: It also includes a step of verifying the degradation effect of the strain.

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