A method for characterizing and isolating gut microbiota capable of specifically uptake or metabolizing natural products.

Abstract

This invention discloses a method for characterizing and isolating gut microbiota capable of specifically uptake or metabolizing natural products, comprising: in vitro co-culture, click chemistry reaction, and flow cytometry sorting. Based on click chemistry technology, this invention co-cultures alkyne-modified natural products with fecal microbiota. Through a copper-catalyzed azido-alkynyl cycloaddition reaction, azidolated fluorescent dyes are labeled onto all bacteria capable of uptake and utilizing natural products or their metabolites. These bacteria are then isolated using flow cytometry sorting. Compared to methods for studying metabolism and utilization using single-species culture, this invention is lower in cost and shorter in time, providing a new approach for extensive and in-depth research on the interaction between gut microbiota and natural products.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and relates to a method for characterizing and isolating gut microbiota that can specifically take up or metabolize natural products; specifically, it relates to a method for characterizing and isolating gut microbiota that can specifically take up or metabolize natural products based on click chemistry technology. Background Technology

[0002] The human gut is home to approximately 10 trillion bacteria, comprising hundreds or thousands of bacterial taxa, which play a vital role in human health. Gut microbiota produce numerous small molecules through primary and secondary metabolic pathways, some of which remain in the gut, while others enter circulation and are chemically modified by the host or excreted in urine. Different gut bacteria specifically absorb different nutrients; for example, Bacteroides, Bifidobacterium, and Lactobacillus primarily absorb and metabolize dietary sphingosine, while Bacteroides, Bifidobacterium, Enterococcus, and Parabacterium are the main interacting microorganisms for dietary cholesterol.

[0003] Natural products, including amino acids, polysaccharides, vitamins, fats, alkaloids, flavonoids, terpenes, phenols, and steroidal compounds, have been shown to prevent and treat a variety of diseases by regulating the balance of the gut microbiota. Natural products not only act on the gut microbiota by inhibiting or promoting the growth of certain bacterial genera and altering its structure, thus exerting their effects, but they are also metabolized and transformed by the gut microbiota through reactions including hydrolysis, deglycosylation, and enzymatic hydrolysis.

[0004] Click chemistry, also known as "link chemistry," is a process that rapidly and reliably synthesizes various molecules by assembling small units. A representative reaction is the copper-catalyzed azido-alkynyl Husigen cycloaddition reaction. Click chemistry reactions are fast, simple to operate, and occur under mild conditions with non-toxic byproducts. Besides chemical synthesis, click chemistry technology has been applied in various fields such as drug target identification, new drug development, cancer tracking, and molecular recognition and sensing.

[0005] A major challenge in studying the uptake and metabolism of natural products by gut microbiota is the vast diversity of gut microbiota. Using traditional single-strain culture methods to screen thousands of isolated strains for their ability to metabolize a particular compound is costly and time-consuming. Furthermore, some strains are difficult to isolate and culture. Therefore, traditional methods cannot encompass all gut microbiota.

[0006] Furthermore, there are differences in gene expression and biochemical transformation processes between monocultured bacterial species and mixed-culture bacterial populations. Compounds that are not metabolized in monocultured bacteria may be taken up and utilized by bacteria in mixed cultures. Therefore, traditional monoculture methods are not suitable for high-throughput screening that simulates the uptake and utilization of compounds by gut microbiota. Summary of the Invention

[0007] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing technologies by providing a method for characterizing and isolating gut microbiota that can specifically take up or metabolize natural products, thus providing new insights for studying the interaction between natural products and gut microbes.

[0008] This invention provides a rapid method for characterizing and isolating bacteria capable of specifically taking up or metabolizing a natural product, based on click chemistry. It comprises three steps: in vitro co-culture, click chemistry reaction, and flow cytometry sorting. Compared to methods for studying metabolism and utilization using single-strain cultures, this method is lower in cost and shorter in time.

[0009] This invention characterizes and isolates gut microbiota that specifically take up or metabolize natural products based on the alkyne-azide cycloaddition reaction, a representative reaction in click chemistry. The natural product structure is terminally modified with an alkyne group, and the fluorescent dye AlexaFluor 647 is terminally modified with an azide. After bacteria take up the alkyne-modified natural product or a natural product metabolite retaining the alkyne group, they undergo a click chemistry reaction with the subsequently added AF647 azide, thus becoming labeled with red fluorescence. AF647 is isolated using flow cytometry. + A bacterial community, that is, a group of bacteria that can specifically take up natural products.

[0010] Technical solution: The objective of this invention is achieved through the following technical solution:

[0011] This invention provides a method for characterizing and isolating gut microbiota capable of specifically uptake or metabolizing natural products, comprising the following steps:

[0012] (1) In vitro co-culture: The microbial community and the natural product are co-cultured in a culture medium;

[0013] (2) Click chemical reaction: The bacteria cultured in step (1) are subjected to a copper-catalyzed azide-alkyne cycloaddition reaction;

[0014] (3) Flow cytometry sorting: Bacteria that take up natural products or metabolites of natural products are labeled with azide-treated fluorescent dyes and collected by a flow cytometer to collect bacteria that can take up natural products and those that cannot.

[0015] The present invention provides a method for characterizing and isolating gut microbiota that preferably specifically uptake or metabolize natural products, comprising the following steps:

[0016] (1) The microbial community and natural products were co-cultured in liquid culture medium and divided into: blank control group, unmodified natural product group and alkynyl modified natural product group.

[0017] (2) Collect bacterial precipitate, fix and permeate bacterial cells, and then perform click chemical reaction with azide fluorescent dye;

[0018] (3) Using a flow cytometer, the areas where fluorescent signal negative bacterial populations were located were circled according to the blank control group and the unmodified natural product group, and the areas where fluorescent signal positive bacterial populations were circled according to the alkyne-modified natural product group; the cell sorting parameters were adjusted to collect negative and positive bacterial populations.

[0019] More preferably, the microbial community is fecal microbiota; the natural product is selected from any one of curcumin, ergosterol, myristic acid, taurine, resveratrol, puerarin, tripterygium lactone, baicalin, naringenin, or quercetin.

[0020] Further, in step (1), the culture time is 6–48 h, the concentration of the natural product is 20–500 μM, and the culture medium components, per liter, include: 5.0–10.0 g peptone, 1.0–3.5 g soybean peptone, 4.0–6.0 g peptone, 8.0–11.0 g serum digest powder, 2.0–3.0 g beef extract powder, 2.0–3.0 g yeast extract powder, 1.0–2.0 g liver extract powder, 5.0–6.0 g soluble starch, 0.5–1.0 g glucose, 1.0–3.0 g sodium chloride, 2.5–3.0 g potassium dihydrogen phosphate, 0.2–1.0 g L-tryptophan, 0.2–1.0 g L-arginine, and 0.3–1.0 g… L-cysteine ​​hydrochloride, 0.3–1.0 g sodium thioglycolate, 0.001–0.01 g heme chloride, 0.001–0.01 g vitamin K1.

[0021] Further, in step (2), the method for collecting bacterial precipitate is as follows: centrifuge at 18000g for 5-10 min to collect bacterial precipitate, add 1mL of 1% BSA / PBS, and wash three times.

[0022] Further, in step (2), the method for fixing the cells and bacteria is as follows: add 0.5-1 mL of 4% paraformaldehyde fixative to the bacterial precipitate, fix it at room temperature for 10-20 min, and then wash it once.

[0023] Further, in step (2), the permeation method is as follows: add 0.5-1 mL of Triton X-100 immunostaining permeation solution, incubate at room temperature for 20-30 min, wash and resuspend in 100-300 μL of 1% BSA / PBS for later use.

[0024] Further, in step (2), the azide fluorescent dye is Alexa Fluor 647 azide.

[0025] Further, in step (2), the click chemistry reaction method is as follows: using a Click Chemistry Reaction Buffer Kit, add 109 μL of reaction buffer (Reaction Buffer A) and 1 μL of azide fluorescent dye to 50 μL of bacterial culture, with the final concentration of the azide fluorescent dye being 1-10 μM, and briefly vortex to mix evenly; add 20 μL of additive (Additive 1), and briefly vortex to mix evenly; add 20 μL of copper sulfate (Copper(II) Sulfate), and briefly vortex to mix evenly; add 10 μL of reducing agent, and briefly vortex to mix evenly, and start the click chemistry reaction, incubating at room temperature in the dark for 30-60 min; wash 3-5 times with 1 mL of 1% BSA / PBS, and resuspend the bacterial pellet in 1-1.5 mL of sterile PBS for later use.

[0026] Further, in step (3), the cell sorting parameters are: nozzle: 70 μm, frequency: 85-90 kHz, amplitude: 5.0-5.5 V, droplet delay: 47.0-47.5, and sheath fluid pressure: 65-75 psi.

[0027] Beneficial effects:

[0028] This invention applies click chemistry technology to study the uptake and utilization of natural products by gut microbiota. Flow cytometry sorting using the AF647 fluorescent dye in the click chemistry reaction can separate bacteria that take up or metabolize natural products. First, this invention demonstrates that the natural products before and after alkyne modification have no significant effect on fecal microbiota or the state of the culture medium. Then, bacteria co-cultured with alkyne-modified natural products undergo a copper-catalyzed azide-alkyne cycloaddition reaction. All bacteria that take up natural products or their metabolites are labeled with the azide-modified fluorescent dye. Flow cytometry sorting is used to collect bacteria that can and cannot take up natural products. Compared with single-species culture methods for studying metabolism and utilization, this invention is lower in cost and shorter in time, providing a new approach for extensive and in-depth research on the interaction between gut microbiota and natural products. Attached Figure Description

[0029] Figure 1 The structural formula of the natural product is shown; among which... Figure 1 A represents curcumin. Figure 1 B is curcuminine. Figure 1 C stands for myristic acid. Figure 1 D is myristic acid alkyne;

[0030] Figure 2 This is a graph showing the pH changes in the culture medium after co-culturing natural products with fecal microbiota; among which... Figure 2 A represents the curcumin and curcuminyne group. Figure 2 B belongs to the myristic acid and myristic acid alkyne group;

[0031] Figure 3 This graph shows the changes in short-chain fatty acid content after co-culturing natural products with fecal microbiota. Figure 3 A represents the change in acetic acid content in the curcumin and curcuminyne groups; Figure 3 B represents the change in propionic acid content in the curcumin and curcumin-acetylenic groups; Figure 3 C represents the change in acetic acid content in the myristic acid and myristic acid alkyne groups; Figure 3 D represents the change in propionic acid content in the myristic acid and myristic acid-alkynyl groups;

[0032] Figure 4 This is a gating diagram for flow cytometry cell sorting. Figure 4 A is sterile PBS. Figure 4 B is the control group. Figure 4 C represents the curcumin group. Figure 4 D belongs to the myristic acid group. Figure 4 E represents the curcuminyn group. Figure 4 F represents the myristoylene group. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0034] Example 1: Characterization and isolation of gut microbiota capable of specifically uptake or metabolize curcumin

[0035] (1) Effects of curcumin and curcuminine on pH of fecal microbiota culture medium

[0036] The structural formulas of curcumin and curcuminyne are shown below. Figure 1 .

[0037] Fresh stool samples were collected from three healthy volunteers who had not taken antibiotics within the past three months. The collected stool samples were stored in sterile centrifuge tubes and processed immediately. The three stool samples were mixed at a mass ratio of 1:1:1 and a 10% stool suspension was prepared using a stool diluent (containing 0.24 g KH₂PO₄, 1.44 g Na₂HPO₄, 8.0 g NaCl, and 0.2 g KCl per liter). After vortexing, the suspension was centrifuged at 3000 rpm for 5 minutes, and the supernatant (fecal microbial suspension) was collected for later use.

[0038] Add 8.99 mL of culture medium (containing 5.0 g peptone, 3.0 g soybean peptone, 5.0 g succinate, 10.0 g serum digest powder, 2.2 g beef extract, 2.5 g yeast extract, 1.2 g liver extract, 5.0 g soluble starch, 0.5 g glucose, 3.0 g sodium chloride, 2.5 g potassium dihydrogen phosphate, 0.2 g L-tryptophan, 1.0 g L-arginine, and 0.3 g glucose per liter) to a 10 mL centrifuge tube. L-cysteine ​​hydrochloride, 0.3 g sodium thioglycolate, 0.005 g heme chloride, 0.001 g vitamin K1), 1 mL fecal microbial solution, and 10 μL of filtered sterilized curcumin (Cur) or curcuminine acetylene (CurA) (final concentration 25 μM) were added to the control group. 10 μL of anhydrous ethanol was added, mixed thoroughly, sealed, and rapidly transferred to a 37°C anaerobic incubator. The mixture was incubated for 48 h, and samples were collected at 0, 6, 12, 24, and 48 h to detect pH changes. Figure 2 As shown in A, there is no significant difference in the effect of Cur and CurA on the pH of the culture medium, indicating that CurA will not affect the pH of the fecal bacteria culture medium due to alkyne modification.

[0039] (2) Effects of Cur and CurA on the content of short-chain fatty acids produced by fecal microbiota

[0040] Culture media collected at 6, 12, 24, and 48 h from the Control, Cur, and CurA groups were respectively incubated with 0.6 mL of 50% H2SO4 and 4 mL of diethyl ether on ice for 20 min, centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and stored on ice for the determination of short-chain fatty acid content using SHIMADZU GC-2014 gas chromatography.

[0041] The chromatographic conditions were as follows: column: SH-Rtx-5 (30cm×0.25mm×0.25μm), carrier gas: N2, flow rate: 21.9mL / min, split ratio: 50, injection port temperature: 200℃, detector temperature: 240℃, injection volume: 1μL, and analysis time: 26min.

[0042] Depend on Figure 3 A and Figure 3 As shown in B, Cur and CurA did not show a significant difference in the production of short-chain fatty acids in fecal microbiota, indicating that CurA would not affect the production of short-chain fatty acids in fecal microbiota due to alkynyl modification.

[0043] (3) Copper-catalyzed azide-alkyne cycloaddition staining

[0044] Bacterial pellets from the 24-hour culture media of the Control, Cur, and CurA groups were collected by centrifugation at 18000g for 10 min. Each pellet was washed three times with 1 mL of 1% BSA / PBS. The bacterial pellets were then fixed with 1 mL of 4% paraformaldehyde fixative at room temperature for 10 min, followed by a gentle wash with 1 mL of 1% BSA / PBS. 1 mL of immunostaining permeabilization buffer (Triton X-100, Beyotime) was added, and the pellets were incubated at room temperature for 30 min. After washing with 1 mL of 1% BSA / PBS, the pellets were resuspended in 300 μL of 1% BSA / PBS for later use (this is the bacterial suspension).

[0045] According to the instructions of the Click Chemistry Reaction Buffer Kit (ClickChemistry Tools), add 109 μL of Reaction Buffer A (the kit includes Reaction Buffer, Additive 1, Reducing Agent, and Copper(II) Sulfate) and 1 μL of AF647-azide (Invitrogen) to 5 μM of bacterial culture. Briefly vortex to mix. Add 20 μL of Additive 1 and briefly vortex to mix. Add 20 μL of Copper(II) Sulfate and briefly vortex to mix. Add 10 μL of Reducing Agent and briefly vortex to mix. Start the click chemistry reaction and incubate at room temperature in the dark for 45 min. Wash five times with 1 mL of 1% BSA / PBS, and resuspend the bacterial pellet in 1 mL of sterile PBS for later use (this is the bacterial suspension sample).

[0046] (4) Bacterial sorting based on AF647

[0047] For flow cytometry (FACSAriaⅢ, BD Biosciences), sterile 1×PBS was used for the sheath fluid, and bacterial suspension samples were filtered through a 400-mesh nylon cell filter before analysis. AF647-azide fluorescent dye was excited using a 633nm red laser, and the fluorescence signal was captured using a 660nm / 20nm filter.

[0048] First, the signal in the solvent PBS is detected to ensure that there are no debris interferences in the solvent background, such as... Figure 4 As shown in Figure A.

[0049] 10,000 particles were collected during sample injection. Fragments were excluded using forward scatter area (FSC-A) and side scatter area (SSC-A), and adherent bacterial cells were excluded using FSC-A and forward scatter height (FSC-H). The AF647 signal intensity was then determined based on the negative control group (Control) and the Cur group within the range of 0-10. 2 The negative gate P3, such as Figure 4 B and Figure 4 As shown in C.

[0050] This gate was then applied to group CurA, and the AF647 signal strength was circled to be greater than 10. 2 Positive gate P4, such as Figure 4 As shown in E.

[0051] CurA-AF647 + 82.9% of the total, CurA-AF647 - The proportion was 15.3%. Cell sorting parameters were adjusted as follows: nozzle: 70 μm, frequency: 87 kHz, amplitude: 5.3 V, droplet delay: 47.45, sheath fluid pressure: 70 psi. AF647 cells were collected using two sterile 1.5 mL centrifuge tubes with the caps cut off. + and AF647 - A community of bacteria.

[0052] Example 2: Characterization and isolation of gut microbiota capable of specifically taking up or metabolizing myristic acid

[0053] (1) Effects of myristic acid and myristic acid on pH of fecal microbiota culture medium

[0054] The structural formulas of myristic acid and myristic acid alkyne are shown below. Figure 1 .

[0055] Fresh stool samples were collected from three healthy volunteers who had not taken antibiotics within the past three months. The stool samples were collected, stored in sterile centrifuge tubes, and processed immediately. The three stool samples were mixed at a mass ratio of 1:1:1 and a 10% stool suspension was prepared using a stool diluent (containing 0.24 g KH₂PO₄, 1.44 g Na₂HPO₄, 8.0 g NaCl, and 0.2 g KCl per liter). After vortexing, the suspension was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected for later use.

[0056] Add 8.99 mL of culture medium (containing 5.0 g peptone, 3.0 g soybean peptone, 5.0 g succinate, 10.0 g serum digest powder, 2.2 g beef extract, 2.5 g yeast extract, 1.2 g liver extract, 5.0 g soluble starch, 0.5 g glucose, 3.0 g sodium chloride, 2.5 g potassium dihydrogen phosphate, 0.2 g L-tryptophan, 1.0 g L-arginine, and 0.3 g glucose per liter) to a 10 mL centrifuge tube. L-cysteine ​​hydrochloride, 0.3 g sodium thioglycolate, 0.005 g heme chloride, 0.001 g vitamin K1), 1 mL fecal microbial solution, and 10 μL of filtered sterilized myristic acid (MA) or myristicoylene (MAA) (final concentration 25 μM) were added to the control group. 10 μL of anhydrous ethanol was added, mixed thoroughly, sealed, and rapidly transferred to a 37°C anaerobic incubator. The mixture was incubated for 48 h, and samples were collected at 0, 6, 12, 24, and 48 h to detect pH changes. Figure 2 As shown in B, there is no significant difference in the effect of MA and MAA on the pH of the culture medium, indicating that MAA will not affect the pH of the fecal bacteria culture medium due to alkyne modification.

[0057] (2) Effects of MA and MAA on the content of short-chain fatty acids produced by fecal microbiota

[0058] Culture media collected at 6, 12, 24, and 48 h from the Control, MA, and MAA groups were added to 4 mL of each medium with 0.6 mL of 50% H2SO4 and 4 mL of diethyl ether. The medium was incubated on ice for 20 min, centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and stored on ice for the determination of short-chain fatty acid content using SHIMADZU GC-2014 gas chromatography.

[0059] The chromatographic conditions were as follows: column: SH-Rtx-5 (30cm×0.25mm×0.25μm), carrier gas: N2, flow rate: 21.9mL / min, split ratio: 50, injection port temperature: 200℃, detector temperature: 240℃, injection volume: 1μL, and analysis time: 26min.

[0060] Depend on Figure 3 C and Figure 3 As shown in D, there is no significant difference between MA and MAA in the production of short-chain fatty acids in fecal microbiota, indicating that MAA does not affect the production of short-chain fatty acids in fecal microbiota due to alkynyl modification.

[0061] (3) Copper-catalyzed azide-alkyne cycloaddition staining

[0062] Bacterial pellets from the 24-hour culture media of the Control, MA, and MAA groups were collected by centrifugation at 18000g for 10 min. Each pellet was washed three times with 1 mL of 1% BSA / PBS. The bacterial pellets were then fixed with 1 mL of 4% paraformaldehyde fixative at room temperature for 10 min, followed by a gentle wash with 1 mL of 1% BSA / PBS. 1 mL of immunostaining permeabilization buffer (Triton X-100, Beyotime) was added, and the pellets were incubated at room temperature for 30 min. After washing with 1 mL of 1% BSA / PBS, the pellets were resuspended in 300 μL of 1% BSA / PBS for later use (this is the bacterial suspension).

[0063] According to the instructions of the Click Chemistry Reaction Buffer Kit (ClickChemistry Tools), add 109 μL of Reaction Buffer A and 1 μL of AF647-azide (Invitrogen) to 5 μM of bacterial culture, and briefly vortex to mix. Add 20 μL of Additive 1 and briefly vortex to mix. Add 20 μL of Copper(II) Sulfate and briefly vortex to mix. Add 10 μL of Reducing Agent and briefly vortex to mix. Start the click chemistry reaction and incubate at room temperature in the dark for 45 min. Wash five times with 1 mL of 1% BSA / PBS, and resuspend the bacterial pellet in 1 mL of sterile PBS for later use (this is the bacterial suspension sample).

[0064] (4) Bacterial sorting based on AF647

[0065] For flow cytometry (FACSAriaⅢ, BD Biosciences), sterile 1×PBS was used for the sheath fluid, and bacterial suspension samples were filtered through a 400-mesh nylon cell filter before analysis. AF647-azide fluorescent dye was excited using a 633nm red laser, and the fluorescence signal was captured using a 660nm / 20nm filter.

[0066] 10,000 particles were collected during injection. Fragments were excluded using FCS-A and SSC-A, and adherent bacterial cells were excluded using FSC-A and FSC-H. The AF647 signal intensity was then determined based on the negative control group (Control group) and the MA group within the range of 0-10. 2 The negative gate P3, such as Figure 4 B and Figure 4 As shown in D.

[0067] This gate is then applied to the MAA group, highlighting AF647 signals with a strength greater than 10. 2Positive gate P4, such as Figure 4 As shown in F.

[0068] MAA-AF647 + 23.6% of the total, MAA-AF647 - The proportion was 74.6%. Cell sorting parameters were adjusted as follows: nozzle: 70 μm, frequency: 87 kHz, amplitude: 5.3 V, droplet delay: 47.45, sheath fluid pressure: 70 psi. AF647 cells were collected using two sterile 1.5 mL centrifuge tubes with the caps cut off. + and AF647 - A community of bacteria.

[0069] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for characterizing and isolating a gut microbiota that can specifically take up or metabolize a natural product, characterized in that, Comprising the following steps: (1) In vitro co-culture: co-culture the flora with natural products in liquid medium, and divide into groups: blank control group, unmodified natural product group and alkynyl modified natural product group; (2) Collect bacterial precipitate, fix and permeabilize the bacteria, and then perform click chemistry reaction with azide fluorescent dye: perform copper-catalyzed azide-alkyne cycloaddition reaction on the bacteria cultured in step (1); (3) Flow cytometry sorting: bacteria that have taken up natural products or natural product metabolites are labeled with azidated fluorescent dye, and the region where the fluorescent signal negative bacterial population is located is circled according to the blank control group and the unmodified natural product group, and the region where the fluorescent signal positive bacterial population is located is circled according to the alkynyl modified natural product group; adjust the cell sorting parameters, and collect the negative and positive bacterial populations, i.e. collect bacteria that can and cannot take up natural products; The natural product is curcumin or myristic acid.

2. The method of characterisation and isolation of a gut community that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, The flora is fecal flora.

3. The method of characterisation and isolation of a gut community that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, In step (1), the culture time is 6-48 h, the natural product concentration is 20-500 μM, and the culture medium components per liter include: 5.0-10.0 g peptone, 1.0-3.5 g soybean peptone, 4.0-6.0 g tryptone, 8.0-11.0 g serum digest powder, 2.0-3.0 g beef infusion powder, 2.0-3.0 g yeast infusion powder, 1.0-2.0 g liver infusion powder, 5.0-6.0 g soluble starch, 0.5-1.0 g glucose, 1.0-3.0 g sodium chloride, 2.5-3.0 g potassium dihydrogen phosphate, 0.2-1.0 g L-tryptophan, 0.2-1.0 g L-arginine, 0.3-1.0 g L-cysteine hydrochloride, 0.3-1.0 g sodium thioglycolate, 0.001-0.01 g hematin chloride, and 0.001-0.01 g vitamin K1.

4. The method of characterisation and isolation of a gut community that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, In step (2), the method for collecting bacterial precipitate is: centrifuge at 18000 g for 5-10 min to collect bacterial precipitate, and wash three times with 1 mL of 1% BSA / PBS.

5. The method of characterisation and isolation of a gut community that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, In step (2), the bacterial fixation method is: add 0.5-1 mL of 4% paraformaldehyde fixing solution to the bacterial precipitate, fix at room temperature for 10-20 min, and then wash once.

6. The method of characterisation and isolation of a gut community that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, In step (2), the permeabilization method is: add 0.5-1 mL of immunostaining permeabilization solution Triton X-100, incubate at room temperature for 20-30 min, and then resuspend in 100-300 μL of 1% BSA / PBS for standby.

7. The method of characterisation and isolation of a gut microbiota that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, In step (2), the azide fluorescent dye is Alexa Fluor 647 azide.

8. The method of characterisation and isolation of a gut community that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, In step (2), the click chemistry reaction method is as follows: 50 μL of bacterial solution is added into 109 μL of reaction buffer Reaction Buffer A and 1 μL of azide fluorescent dye, the final concentration of azide fluorescent dye is 1-10 μM, and the mixture is briefly vortexed and mixed uniformly; 20 μL of additive Additive 1 is added, the mixture is briefly vortexed and mixed uniformly; 20 μL of copper (II) sulfate is added, the mixture is briefly vortexed and mixed uniformly; 10 μL of reducing agent Reducing Agent is added, the mixture is briefly vortexed and mixed uniformly, the click chemistry reaction is started, and incubation is performed at room temperature in the dark for 30-60 min; 1 mL of 1% BSA / PBS is used for washing 3-5 times, and the bacterial precipitate is resuspended in 1-1.5 mL of sterile PBS for standby.

9. The method of characterisation and isolation of a gut microbiota that specifically uptakes or metabolises a natural product according to claim 1, characterised in that, In step (3), the cell sorting parameters are as follows: nozzle: 70 μm, frequency: 85-90 kHz, amplitude: 5.0-5.5 V, droplet delay: 47.0-47.5, and sheath fluid pressure: 65-75 psi.

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