Estrogen combined degradation system of non-antagonistic functional bacteria and its preparation method and application

Abstract

The invention relates to the technical field of microorganisms, and in particular to an estrogen combined degradation system of non-antagonistic functional bacterial communities, a preparation method and an application thereof; the combined degradation system is composed of four strains that do not have antagonistic effects on each other: Neosphingobacterium ES2-1, Rhodococcus JX-2, Sphingomonas E2S and Neosphingobacterium ARI-1; the functional bacterial communities composed of the four bacteria can construct a combined degradation system for natural estrogens based on a quorum sensing mechanism between strains; when stimulated by natural estrogens, the combined degradation system can not only promote the dehydrogenation reaction of the D ring in the initial stage of degradation of estriol and estradiol, but also stimulate the expression of related proteins involved in the synergistic transport ability and the electron transfer ability in the strains, thereby making the degradation effect of the functional bacterial community on natural estrogens significantly higher than any other permutation and combination except the combination of the functional bacterial species.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an estrogen combined degradation system of a non-antagonistic functional bacterial community and a preparation method and application thereof. Background Art

[0002] Environmental estrogens are divided into natural estrogens and synthetic estrogens. Natural estrogens are more common and are mainly derived from livestock and poultry manure and human feces and urine. When estrogens enter rivers, groundwater, soil and crops with precipitation and surface runoff, they will threaten ecological security and human health.

[0003] Natural estrogens include estriol (E3), estradiol (E2) and estrone (E1), among which estradiol has the strongest estrogenic activity, followed by estrone, and estriol is the weakest. During metabolism in the body, estrone and estriol are both degradation products of estradiol. Under certain conditions, the three will transform. When they cannot be metabolized in time, estriol will often accumulate. Therefore, degrading only one or two of the estrogens is of little significance in practical applications. More often, it is necessary to degrade all three estrogens simultaneously.

[0004] Biotechnology has attracted much attention because of its green and environmentally friendly nature and its significant effect on the degradation of estrogen. For example, the article "Pathway and Molecular Mechanism of 17β-estradiol Metabolism by Estrogen-Degrading Bacteria Novosphingobium sp.ES2-1" provides a detailed analysis and record of Novosphingobium sp.ES2-1's ability to degrade estradiol.

[0005] Based on the above inspiration, the inventors hope to further improve the degradation rate of estrogen by the strain based on the previous work. During the research process, the inventors discovered a combined degradation system based on the quorum sensing mechanism of bacterial flora. This combined degradation system shows excellent targeted degradation of natural estrogen under specific combinations and application environments. Summary of the invention

[0006] In order to achieve the above-mentioned purpose, the present invention provides a combined degradation system of estrogen with non-antagonistic functional flora, as well as a preparation method and application. The combined degradation system is composed of four strains that do not have antagonistic effects on each other: Neosphingobacterium ES2-1, Rhodococcus JX-2, Sphingomonas E2S, and Neosphingobacterium ARI-1; the functional flora composed of the above four strains can construct a combined degradation system for natural estrogens based on the quorum sensing mechanism between strains, and the combined degradation system can not only promote the dehydrogenation reaction of the D ring in the early stage of estriol and estradiol degradation, but also stimulate the expression of related proteins involved in the co-transport ability and electron transfer ability in the strain.

[0007] It is worth noting that the four strains ES2-1, JX-2, E2S and ARI-1 involved in the scheme of the present invention were discovered by predecessors, and their genetic composition and corresponding functions have been revealed or partially revealed, and are not proposed for the first time by the present invention, that is, the present invention does not protect the strains themselves.

[0008] The present invention protects a functional bacterial community composed of four known strains and the combined degradation ability they exhibit, which is not exhibited in any one of the above four strains, or in a combination of any two or any three of them, and the combined degradation ability has a significant degradation effect on natural estrogen. The above discovery is proposed for the first time in this application.

[0009] Based on the above reasons, the present application intends to protect a functional bacterial flora composed of four known strains, as well as a preparation method of the functional bacterial flora and the optimal applicable conditions for degrading natural estrogen.

[0010] The present invention designs a non-antagonistic functional bacterial community estrogen combined degradation system, the combined degradation system includes a functional bacterial community and a synergistic system;

[0011] The functional bacterial community consists of a lead strain and a partner strain that have no antagonistic relationship with each other, and all strains are deposited by the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University;

[0012] The lead strain is Novosphingobium sp.ES2-1, a Gram-negative bacterium, recorded as ES2-1; ES2-1 was isolated and preserved from activated sludge by Li Shunyao from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University in 2020. It has the function of degrading estriol, estradiol, and estrone, with degradation rates of estriol, estradiol, and estrone of 89.38%, 94.4%, and 82.36% respectively;

[0013] Partner strains include:

[0014] Rhodococcus sp. JX-2, a Gram-positive bacterium, is recorded as JX-2; JX-2 was isolated and preserved from activated sludge by Liu Jingxian from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University in 2014. It has the function of degrading estradiol, with an estradiol degradation rate of 93.06%;

[0015] Sphingomonas sp.E2S, a Gram-negative bacterium, is recorded as E2S. E2S was isolated and preserved from activated sludge by Sun Minxia from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University in 2015. It has the function of degrading estradiol, with an estradiol degradation rate of 63.4%.

[0016] Novosphingobium sp.ARI-1, a Gram-negative bacterium, is recorded as ARI-1; ARI-1 was purchased and preserved by Liu Juan from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University in 2014 from the American Type Culture Collection (ATCC), and has the function of degrading estradiol and estrone, with degradation rates of estradiol and estrone of 89.42% and 77.67% respectively;

[0017] Functional flora:

[0018] The leading strain can degrade the refractory pollutants and the downstream decomposition products of the refractory pollutants: easily degradable pollutants; and the metabolites of the leading strain can accelerate the start-up rate of the partner strain;

[0019] The partner strain can degrade easily degradable pollutants, reducing the degradation burden of the lead strain on easily degradable pollutants, thereby promoting the degradation ability of the lead strain on difficult-to-degrade pollutants;

[0020] The synergistic system includes the signal molecule set of the functional bacterial community and the promoting relationship between the signal molecule set and the expression of the target gene;

[0021] The signal molecule collection includes: acyl homoserine lactone AHL produced by ES2-1, E2S and ARI-1, autoinducing peptide AIP produced by JX-2, and signal molecule AI-2 produced based on quorum sensing of Gram-positive and Gram-negative bacteria;

[0022] When the functional bacterial community is stimulated by refractory or easily degradable pollutants, the lead strain and the partner strain communicate with each other through a set of signal molecules, thereby promoting the expression of the following genes in ES2-1:

[0023] The expression gene of 17β-hydroxysteroid dehydrogenase involved in dehydrogenation ability;

[0024] The expression genes of the ABC transporter encoded by KHS43222.1, the transmembrane protein encoded by WP_039334026.1, and the ATP binding subunit encoded by WP_039331930.1 involved in the co-transport ability;

[0025] The expressed genes involved in electron transfer ability include ATPase encoded by WP_039331930.1 and flavin oxidoreductase encoded by WP_039333259.1.

[0026] The present invention also provides a method for preparing a non-antagonistic functional bacterial community estrogen combined degradation system, the steps comprising:

[0027] S1. Activate and culture the strains ES2-1, JX-2, E2S, and ARI-1 separately:

[0028] The activation culture of S1-1, JX-2, and E2S was performed in LB medium, and the activation culture of ES2-1 and ARI-1 was performed in R2A medium;

[0029] S1-2, set the culture conditions as follows: temperature of 30°C, rotation speed of 150 rpm, initial pH of 7.0, and shading throughout the process;

[0030] S1-3, culturing the bacteria according to the culture conditions in S1-2 until the test strain reaches the logarithmic phase;

[0031] S2, centrifuge the mixed bacterial flora in S1 at 7500-8000 r / min for 5-10 min, remove the supernatant, add an equal amount of MSM medium, resuspend, and centrifuge again at 7500-8000 r / min for 5-10 min; repeat the above steps 2-3 times, collect the last bacterial cells and adjust them with MSM medium to obtain the bacterial solution of each single strain;

[0032] S3. Mix the ES2-1, JX-2, E2S, and ARI-1 bacterial cultures cultured in S2 at a volume ratio of 1:1:1:1 to obtain a functional bacterial flora.

[0033] The present invention also provides the application of the estrogen combined degradation system of non-antagonistic functional bacteria, and the combined degradation system is used to degrade natural estrogen, which includes estriol as a difficult-to-degrade pollutant and estradiol and estrone as both easily degradable pollutants.

[0034] In the present invention, there is no mutual antagonism between the four selected strains, which has been verified by the cross-line method and the filter paper method. In the present invention, ES2-1 can first degrade the estriol in the system into estradiol and estrone or one of the two, and can also degrade the estradiol and estrone in the system synchronously, and as the leading strain, it plays a major role in the efficiency of the three estrogens in the system. In the present invention, JX-2 and E2S can degrade the estradiol originally present in the system or the estradiol produced by metabolism, avoid the accumulation of estradiol, and improve the system's treatment efficiency of estradiol. In the present invention, ARI-1 can degrade the estradiol and estrone originally present in the system or the estradiol and estrone produced by metabolism, avoid the accumulation of estradiol and estrone, and improve the system's treatment efficiency of estradiol and estrone.

[0035] Under the stress of different types of estrogens, the functional bacterial communities produce a synergistic interaction effect. The acyl homoserine lactone AHL produced by ES2-1, E2S and ARI-1, the autoinducing peptide AIP produced by JX-2 and the signal molecule AI-2 produced based on quorum sensing of Gram-positive bacteria and Gram-negative bacteria act as a communication "language". Based on the quorum sensing mechanism, they can increase the expression of 17β-hydroxysteroid dehydrogenase in ES2-1 and promote the strain's co-transport function and electron transfer function, thereby improving the degradation of natural estrogens by the entire system.

[0036] Under the following laboratory culture conditions: when pH = 7.0, temperature 30°C, inoculation size 5%, initial concentration 5 mg / L, the functional bacterial community can achieve degradation rates of 97.21%, 99.62% and 99.07% for E1, E2 and E3 with an initial concentration of 20 mg / L, respectively, within 7 days; the results show that the functional bacterial community has a significantly higher degradation effect on natural estrogen than any strain that constitutes the bacterial community.

[0037] Compared with the existing natural estrogen degradation method, the beneficial effects of the present invention are:

[0038] (1) The present invention provides a functional bacterial community composed of four strains that do not have antagonistic effects on each other. The functional bacterial community can construct a joint degradation system for natural estrogens based on the quorum sensing mechanism between the strains. When stimulated by natural estrogens, the joint degradation system can not only promote the dehydrogenation reaction of the D ring in the early stage of estriol and estradiol degradation, but also promote the expression of related proteins involved in the co-transport ability and electron transfer ability in the strain.

[0039] (2) Under the following laboratory culture conditions: pH = 7.0, temperature 30°C, inoculation size 5%, initial concentration 5 mg / L, the functional bacterial community can degrade E1, E2 and E3 with an initial concentration of 20 mg / L by 97.21%, 99.62% and 99.07% respectively within 7 days; the results show that the degradation effect of the functional bacteria on natural estrogen is significantly higher than that of any other combinations except the combination of the functional bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The effects of different environmental factors on the degradation of natural estrogen by functional bacteria, where (a) is the initial concentration of estrogen, (b) is the inoculation amount, (c) is the temperature, and (d) is the pH value;

[0041] Figure 2 It is the removal effect of single strain and functional flora on natural estrogen. DETAILED DESCRIPTION

[0042] In order to further illustrate the method and effect of the present invention, the technical solution of the present invention will be clearly and completely described in combination with experiments.

[0043] Example 1: This example describes a method for preparing a combined degradation system of non-antagonistic functional bacteria.

[0044] S1. Activate and culture the four strains ES2-1, JX-2, E2S, and ARI-1 separately:

[0045] The activation culture of S1-1, JX-2, and E2S was performed in LB medium, and the activation culture of ES2-1 and ARI-1 was performed in R2A medium;

[0046] S1-2, set the culture conditions as follows: temperature of 30°C, rotation speed of 150 rpm, initial pH of 7.0, and shading throughout the process;

[0047] S1-3, culturing the bacteria according to the culture conditions in S1-2 until the test strain reaches the logarithmic phase;

[0048] S2, centrifuge the mixed bacterial flora in S1 at 7500r / min for 5min, remove the supernatant, add an equal amount of MSM medium, resuspend, and centrifuge again at 7500r / min for 5min; repeat the above steps twice, collect the last bacterial cells and adjust them with MSM medium to obtain the bacterial solution of each single strain;

[0049] S3. Mix the ES2-1, JX-2, E2S, and ARI-1 bacterial cultures cultured in S2 at a volume ratio of 1:1:1:1 to obtain a functional bacterial flora.

[0050] Example 2: This example describes a method for preparing a combined degradation system of non-antagonistic functional bacteria.

[0051] S1. Activate and culture the strains ES2-1, JX-2, E2S, and ARI-1 separately:

[0052] The activation culture of S1-1, JX-2, and E2S was performed in LB medium, and the activation culture of ES2-1 and ARI-1 was performed in R2A medium;

[0053] S1-2, set the culture conditions as follows: temperature of 30°C, rotation speed of 150 rpm, initial pH of 7.0, and shading throughout the process;

[0054] S1-3, culturing the bacteria according to the culture conditions in S1-2 until the test strain reaches the logarithmic phase;

[0055] S2, centrifuge the mixed bacterial flora in S1 at 8000r / min for 10min, remove the supernatant, add an equal amount of MSM medium, resuspend, and centrifuge again at 8000r / min for 10min; repeat the above steps 3 times, collect the last bacterial cells and adjust them with MSM medium to obtain the bacterial solution of each single strain;

[0056] S3. Mix the ES2-1, JX-2, E2S, and ARI-1 bacterial cultures cultured in S2 at a volume ratio of 1:1:1:1 to obtain a functional bacterial flora.

[0057] Example 3: The content of this example is: a non-antagonistic functional bacterial flora prepared based on the preparation method in Example 2.

[0058] The non-antagonistic functional bacterial community joint degradation system designed by the present invention comprises a functional bacterial community and a synergistic system.

[0059] The functional bacterial community is composed of a leading strain and a partner strain that have no antagonistic relationship with each other. All bacterial species are preserved by the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University.

[0060] The lead strain is Novosphingobium sp.ES2-1, a Gram-negative bacterium, recorded as ES2-1; ES2-1 was isolated and preserved from activated sludge by Li Shunyao from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University in 2020. It has the function of degrading estriol, estradiol, and estrone, with degradation rates of estriol, estradiol, and estrone of 89.38%, 94.4%, and 82.36% respectively;

[0061] Partner strains include:

[0062] Rhodococcus sp. JX-2, a Gram-positive bacterium, is recorded as JX-2; JX-2 was isolated and preserved from activated sludge by Liu Jingxian from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University in 2014. It has the function of degrading estradiol, with an estradiol degradation rate of 93.06%;

[0063] Sphingomonas sp.E2S, a Gram-negative bacterium, is recorded as E2S. E2S was isolated and preserved from activated sludge by Sun Minxia from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University in 2015. It has the function of degrading estradiol, with an estradiol degradation rate of 63.4%.

[0064] Novosphingobium sp.ARI-1, a Gram-negative bacterium, is recorded as ARI-1; ARI-1 was purchased and preserved by Liu Juan from the Institute of Soil Organic Pollution Control and Remediation of Nanjing Agricultural University from the American Type Culture Collection (ATCC) in 2014. It has the function of degrading estradiol and estrone, with degradation rates of estradiol and estrone of 89.42% and 77.67% respectively.

[0065] Functional flora:

[0066] The leading strain can degrade the refractory pollutants and the downstream decomposition products of the refractory pollutants: easily degradable pollutants; and the metabolites of the leading strain can accelerate the start-up rate of the partner strain;

[0067] Partner strains can degrade easily degradable pollutants and reduce the degradation burden of the leading strain on easily degradable pollutants, thereby promoting the leading strain's ability to degrade difficult-to-degrade pollutants.

[0068] The synergistic system includes the signal molecule set of the functional bacterial community and the promoting relationship between the signal molecule set and the expression of the target gene;

[0069] The signal molecule collection includes: acyl homoserine lactone AHL produced by ES2-1, E2S and ARI-1, autoinducing peptide AIP produced by JX-2, and signal molecule AI-2 produced based on quorum sensing of Gram-positive and Gram-negative bacteria;

[0070] When the functional bacterial community is stimulated by refractory or easily degradable pollutants, the lead strain and the partner strain communicate with each other through a set of signal molecules, thereby promoting the expression of the following genes in ES2-1:

[0071] The expression gene of 17β-hydroxysteroid dehydrogenase involved in dehydrogenation ability;

[0072] The expression genes of the ABC transporter encoded by KHS43222.1, the transmembrane protein encoded by WP_039334026.1, and the ATP binding subunit encoded by WP_039331930.1 involved in the co-transport ability;

[0073] The expressed genes involved in electron transfer ability include ATPase encoded by WP_039331930.1 and flavin oxidoreductase encoded by WP_039333259.1.

[0074] Experimental Example 1: The narrative basis of this experimental example is the recording scheme in Example 3, which aims to record the antagonism experiment during the strain compounding process.

[0075] Take the four strains of E2S, JX-2, ES2-1, and ARI-1 for the antagonism experiment between strains. This experiment uses the filter paper method. One of the bacterial liquids cultured to the logarithmic phase is spread on the plate, and the other three bacterial liquids are dipped with filter paper and placed on the plate in turn. Culture at 30°C for 2 days, and observe whether there is a transparent antagonistic circle around the filter paper. The presence of an antagonistic circle indicates an antagonistic reaction. The results are shown in Table 1 ("+" represents an antagonistic reaction, and "-" represents no antagonistic reaction). As shown in Table 1, none of the four strains have an antagonistic reaction.

[0076] Table 1 Results of strain antagonistic reactions

[0077]

[0078]

[0079] Experimental Example 2: The narrative basis of this experimental example is the recording scheme in Example 3, which aims to record the degradation effect of functional bacteria on natural estrogen at different initial estrogen concentrations.

[0080] The functional microbial flora cultured in Example 3 was adjusted to OD using MSM medium. 600 =1.0, at 30°C, inoculation size 5%, pH = 7.0, added to MSM medium with natural estrogen concentrations of 0.1, 0.5, 1, 5, and 10 mg / L respectively, and cultured for 7 days, then extracted with the whole bottle, added with an equal volume of methanol, and ultrasonically vibrated for 30 minutes, then filtered through a 0.25 μm filter membrane and placed in a 2 mL brown liquid vial, and finally the contents of estriol, estradiol, and estrone were detected by liquid chromatography. By comparison, the degradation rates of estriol, estradiol, and estrone were 98.47%, 99.33%, and 99.10% under the condition of 5 mg / L / , and the functional bacteria had the best effect on the degradation of natural estrogen under this concentration condition.

[0081] Experimental Example 3: The narrative basis of this experimental example is the recording scheme in Example 3, which aims to record the degradation effect of functional bacteria on natural estrogen at different inoculation amounts.

[0082] The functional microbial flora cultured in Example 3 was adjusted to OD using MSM medium. 600=1.0, and inoculated with 1%, 3%, 5%, 7%, and 10% MSM medium at pH = 7.0, 30°C, and a natural estrogen concentration of 5 mg / L for 7 days, the whole bottle was used for extraction, an equal volume of methanol was added, and after ultrasonic oscillation for 30 minutes, the mixture was filtered through a 0.25 μm filter membrane and placed in a 2 mL brown liquid vial. Finally, the contents of estriol, estradiol, and estrone were detected by liquid chromatography. By comparison, the degradation rates of estriol, estradiol, and estrone were 99.81%, 99.96%, and 99.67% under the condition of 5%, and the functional bacteria had the best effect on the degradation of natural estrogen under this inoculation amount condition.

[0083] Experimental Example 4: The narrative basis of this experimental example is the recording scheme in Example 3, which aims to record the degradation effect of functional bacteria on natural estrogen at different temperatures.

[0084] The functional microbial flora cultured in Example 3 was adjusted to OD using MSM medium. 600 =1.0, under the conditions of pH=7.0, inoculation size 5%, natural estrogen concentration 5 mg / L, added to MSM culture medium at temperatures of 20°C, 30°C, 40°C, and 50°C, and cultured for 7 days, the whole bottle was extracted, an equal volume of methanol was added, and after ultrasonic oscillation for 30 minutes, it was filtered through a 0.25 μm filter membrane and placed in a 2 mL brown liquid vial. Finally, the contents of estriol, estradiol, and estrone were detected by liquid chromatography. By comparison, the degradation rates of estriol, estradiol, and estrone at 20 and 30°C were 99.38%, 99.55%, and 99.42% and 98.85%, 99.64%, and 99.56%, respectively. Under this temperature condition, the functional bacteria had the best effect on the degradation of natural estrogen.

[0085] Experimental Example 5: The narrative basis of this experimental example is the recording scheme in Example 3, which aims to record the degradation effect of functional bacteria on natural estrogen at different pH values.

[0086] The functional microbial flora cultured in Example 3 was adjusted to OD using MSM medium. 600 =1.0, at 30°C, inoculation size 5%, natural estrogen concentration 5 mg / L, added to MSM medium with pH values ​​of 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and cultured for 7 days, then extracted using the whole bottle, added with an equal volume of methanol, and ultrasonically vibrated for 30 min, filtered through a 0.25 μm filter membrane, and placed in a 2 mL brown liquid vial. Finally, the contents of estriol, estradiol, and estrone were detected by liquid chromatography. By comparison, the degradation rates of estriol, estradiol, and estrone were 99.89%, 100%, and 99.67% under the condition of pH = 7.0, and the functional flora had the best effect on the degradation of natural estrogen under this pH condition.

[0087] Experimental Example 6: The description of this experimental example is based on the protocol described in Example 3, and is intended to compare the degradation effect of functional bacterial flora on natural estrogen compared to a single bacterial strain.

[0088] The functional microbial flora and single strain cultured in Example 3 were adjusted to OD using MSM medium. 600 =1.0, added to MSM medium at 30℃, inoculation volume 5%, natural estrogen concentration 20mg / L, pH 7.0, and cultured for 7 days, extracted with the whole bottle, added with an equal volume of methanol, ultrasonically vibrated for 30min, filtered through a 0.25μm filter membrane and placed in a 2mL brown liquid vial, and finally detected the content of estriol, estradiol, and estrone by liquid chromatography. By comparison, it can be seen that under the conditions, only ES2-1 among the four strains has a degradation effect on the three natural estrogens, with the degradation rates of estriol, estradiol, and estrone being 89.38%, 94.4%, and 82.36%, while the degradation rates of estriol, estradiol, and estrone by the functional flora are 99.07%, 99.62%, and 97.21%. In summary, the functional flora has a wider degradation substrate spectrum for estrogen and a more significant degradation effect.

Claims

1. A non-antagonistic functional bacterial flora with combined estrogen degradation effect, characterized in that: The functional bacteria have a synergistic effect; The functional bacterial community is composed of a leading strain and a partner strain that have no antagonistic relationship with each other; The lead strain is Neosphingobacterium ES2-1, denoted as ES2-1; The partner strains include: Rhodococcus JX-2, denoted as JX-2; Sphingomonas E2S, denoted as E2S; Neosphingobacterium ARI-1, denoted as ARI-1; Among the functional bacteria: The leading strain can degrade refractory pollutants and downstream decomposition products of refractory pollutants: easily degradable pollutants; and the metabolites of the leading strain can accelerate the activation rate of the partner strain; The partner strain can degrade easily degradable pollutants, reducing the degradation burden of the lead strain on easily degradable pollutants, thereby promoting the degradation ability of the lead strain on difficult-to-degrade pollutants; The synergistic effect utilizes a collection of signaling molecules and a promoting relationship between the collection of signaling molecules and the expression of target genes; The signal molecule set includes: acyl homoserine lactone AHL produced by ES2-1, E2S and ARI-1, autoinducing peptide AIP produced by JX-2, and signal molecule AI-2 produced based on quorum sensing of Gram-positive bacteria and Gram-negative bacteria; When the functional bacterial community is stimulated by refractory or easily degradable pollutants, the lead strain and the partner strain communicate with each other through a set of signal molecules, thereby promoting the expression of the following genes in ES2-1: The expression gene of 17β-hydroxysteroid dehydrogenase involved in dehydrogenation ability; The expression genes of the ABC transporter encoded by KHS43222.1, the transmembrane protein encoded by WP_039334026.1, and the ATP binding subunit encoded by WP_039331930.1 involved in the co-transport ability; The expression genes of ATPase encoded by WP_039331930.1 and flavin oxidoreductase encoded by WP_039333259.1, which are involved in electron transfer ability; The preparation method of the functional bacterial flora is as follows: S1. Activate and culture the strains ES2-1, JX-2, E2S, and ARI-1 separately: The activation culture of S1-1, JX-2, and E2S was performed in LB medium, and the activation culture of ES2-1 and ARI-1 was performed in R2A medium; S1-2, set the culture conditions as follows: temperature of 30°C, rotation speed of 150 rpm, initial pH of 7.0, and shading throughout the process; S1-3, culturing the strain according to the culture conditions in S1-2 until the test strain reaches the logarithmic growth phase; S2, centrifuge the mixed bacterial colony in S1 at 7500-8000r / min for 5-10min, remove the supernatant, add an equal amount of MSM medium, resuspend, and centrifuge again at 7500-8000r / min for 5-10min; repeat the above steps 2-3 times, collect the last bacterial cell and adjust the OD with MSM medium 600 =1.0, and then the bacterial solution of each single strain was obtained; S3, mixing the bacterial solutions of ES2-1, JX-2, E2S and ARI-1 cultured in S2 at a volume ratio of 1:1:1:1 to obtain a functional bacterial flora; The functional bacterial flora is used to degrade natural estrogens composed of any one or more of estrone, estradiol or estriol.

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