Method for screening flavone transforming bacteria by carbon source stress

Abstract

The invention relates to the technical field of microorganisms, and particularly provides a method for screening flavone-transformed bacteria by carbon source stress, which is characterized in that a carbon source stress environment is constructed, and a target flavone compound is used as a unique carbon source to induce reprogramming of a microbial metabolic pathway, so that the screening probability of efficient flavone-transformed strains is remarkably improved; according to the method, the limitation of a traditional technology is broken through, leap-type improvement of the screening efficiency is achieved, a carbon source stress mechanism drives target microorganisms to be rapidly enriched, the screening period is shortened, the number of candidate strains is increased, and the false positive rate is reduced to 5% or below in combination with microplate reader quantitative detection and HPLC verification; the operation process is simplified, so that the cost is reduced, and the detection accuracy is improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening technology, and specifically relates to a method for screening flavonoid-transforming bacteria under carbon source stress. Background Technology

[0002] Flavonoids, originally a general term for compounds derived from 2-phenylchromone as their backbone, now broadly refer to a series of compounds with a C6-C3-C6 structure, consisting of two benzene rings linked by three carbon atoms. They are a class of naturally occurring organic compounds widely found in plants, possessing various biological activities such as antioxidant, anti-inflammatory, and anti-tumor effects, and have significant applications in medicine, food, and cosmetics. However, the complex structures of natural flavonoids and the low bioavailability of some limit their applications. Microbial transformation can modify the structure of flavonoids, improving their biological activity and bioavailability. Therefore, screening microbial strains with highly efficient flavonoid transformation capabilities is of great importance.

[0003] Currently, screening methods for flavonoid-transforming bacteria mainly include traditional plate screening and shake-flask fermentation screening. Traditional plate screening involves diluting the sample and spreading it on a plate containing flavonoids, then observing the clear zone or color change around the colonies to screen for positive strains. However, this method has low screening efficiency and is difficult to accurately assess the transforming ability of the strains. Shake-flask fermentation screening involves fermenting the initially screened strains in shake flasks, and assessing the transforming ability of the strains by detecting changes in the content of flavonoids in the fermentation broth. While this method can accurately assess the transforming ability of the strains, it is cumbersome, time-consuming, and not suitable for large-scale screening. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for screening flavonoid-transforming bacteria under carbon source stress.

[0005] The first aspect of this invention provides a method for screening flavonoid-transforming bacteria under carbon source stress, comprising the following steps:

[0006] Step 1: Prepare a carbon source stress screening medium, wherein the glucose concentration in the medium is 0-2.5 g / L, and the target flavonoid compound is added as the sole carbon source;

[0007] Step 2: After serially diluting the environmental samples, spread them onto the culture medium;

[0008] Step 3: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the difference in OD values ​​between 0 h of culture and after culture at a wavelength of 250-500 nm, and screen strains with an OD value difference ≥ 0.4 as candidate strains.

[0009] This invention provides a method for screening flavonoid-transforming bacteria under carbon source stress. By constructing a carbon source stress environment with a glucose concentration of 0-2.5 g / L and using the target flavonoid compound as the sole carbon source, the method induces reprogramming of microbial metabolic pathways, significantly improving the screening probability of highly efficient flavonoid-transforming strains. This method overcomes the limitations of traditional techniques, achieving a leapfrog improvement in screening efficiency—the carbon source stress mechanism drives the rapid enrichment of target microorganisms, shortens the screening cycle, increases the number of candidate strains, and, combined with ELISA quantitative detection and HPLC verification, reduces the false positive rate to below 5%. Simultaneously, it possesses significant industrialization value: simplified operation procedures reduce costs and improve detection accuracy.

[0010] Carbon sources are essential nutrients for microbial growth and metabolism, and different microorganisms have varying abilities to utilize them. Under carbon source stress, microbial metabolic pathways change, potentially inducing the expression of certain enzymes related to flavonoid conversion, thereby enhancing their flavonoid conversion capacity. Therefore, using carbon source stress to screen flavonoid-converting bacteria is an effective method.

[0011] Furthermore, the carbon source stress culture medium includes ammonium nitrate 0.5-2 g / L; dipotassium hydrogen phosphate 0.25-1 g / L; magnesium sulfate 0.125-0.5 g / L; flavonoids 0.1-0.5 g / L; pH 6.3-7.0.

[0012] Furthermore, the flavonoid compound is selected from isoliquiritin, hesperidin, or daidzein.

[0013] Furthermore, the OD value detection wavelengths are as follows: 366nm for isoliquiritigenin detection, 283nm for hesperidin detection, and 280-300nm for daidzein detection.

[0014] Furthermore, in step 2, the cells are incubated at 25-35℃ for 3-7 days.

[0015] Furthermore, the strain screening criteria in step 3 include: fungi: OD difference ≥ 0.4 within 0-72h of culture; bacteria: OD difference ≥ 0.4 within 0-48h of culture.

[0016] Furthermore, step 4 is included, which verifies the strain's ability to convert flavonoids by HPLC, with a positive standard being a new product peak or a peak area change of ≥20%.

[0017] Furthermore, the HPLC validation conditions were as follows: C18 column, mobile phase A was 1% formic acid water, mobile phase B was acetonitrile:methanol = 9:1, flow rate was 1.0 mL / min, column temperature was 30-35℃, and detection wavelength was 280 nm or 370 nm.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. This invention provides a method for screening flavonoid-transforming bacteria under carbon source stress. By constructing a carbon source stress environment with a glucose concentration of 0-2.5 g / L and using the target flavonoid compound as the sole carbon source, the method induces reprogramming of microbial metabolic pathways, significantly improving the screening probability of highly efficient flavonoid-transforming strains. This method overcomes the limitations of traditional technologies, achieving a leapfrog improvement in screening efficiency—the carbon source stress mechanism drives the rapid enrichment of target microorganisms, shortens the screening cycle, increases the number of candidate strains, and, combined with ELISA quantitative detection and HPLC verification, reduces the false positive rate to below 5%. Simultaneously, it possesses significant industrialization value: simplified operation procedures reduce costs and improve detection accuracy.

[0020] 2. By designing culture media with different carbon contents, this invention can rapidly enrich target microorganisms, significantly shorten the screening time, and obtain more candidate strains with transformation capabilities, thus significantly improving screening efficiency.

[0021] 3. This invention is simple to operate, low in cost, highly efficient and accurate, suitable for in-depth research in scientific laboratories and large-scale production in enterprises. It helps to accelerate the research and development of new drugs and health products, and has broad application prospects and significant social benefits. Attached Figure Description

[0022] Figure 1 The images show the morphological characteristics of some strains isolated from different culture media in Example 1. Figure 1 In this context, 'a' represents PDA culture medium (soil sample: ALJ031). Figure 1 In this context, 'b' represents TSB culture medium (soil sample: JS018). Figure 1 In this context, 'c' represents PDA culture medium (soil sample: JS018). Figure 1 In this context, 'd' represents Sabouraud dextrose medium (soil sample: JS018). Figure 1 e and Figure 1 In this context, f represents LB medium (soil sample: JS018). Figure 1 The g in the text represents 1 / 5 of Sabouraud medley medium. Figure 1 h in the text refers to Sabouraud dextrose medium (soil sample: ALJ031).

[0023] Figure 2 The images show the morphological characteristics of some strains after purification in Example 1.

[0024] Figure 3 The results of the enzyme-linked immunosorbent assay (ELISA) screening of the soil microorganisms' ability to convert isoliquiritigenin in Example 1 are shown. Figure 3 In the figure, 'a' represents the difference in OD values ​​of fungi in soil sample (JS018) within 0-72 hours. Figure 3 In the figure, b represents the difference in OD values ​​of fungus (JS013) within 0-72 hours. Figure 3In the figure, 'c' represents the difference in OD values ​​of fungi in soil sample (ALJ031) within 0-72 hours. Figure 3 In this context, d represents the OD difference between the four soil samples and the actinomycetes within 0-48 hours.

[0025] Figure 4 The image shows the liquid chromatogram of the bacterial culture in Example 1 after 7 days of culture at 280 nm. Figure 4 In the figure, 'a' represents the chromatogram of a mixture of Sabouraud broth and standard culture medium. Figure 4 In the figure, b is the chromatogram of the mixed bacterial solution of carbon source stress culture medium and standard.

[0026] Figure 5 A graph showing the difference in OD values ​​of bacteria in a TSB liquid culture medium with a hesperidin concentration of 0.4 mg / mL. Figure 5 Strains 1-31 are listed in sequence; the total volume of the standard was adjusted to a concentration of 0.4 mg / mL, and the OD value represents the difference between the OD values ​​of the hesperidin-containing Sabouraud broth and the strain cultured for 48 h minus 0 h. 1 is 35150; 2 is 35151; 3 is 35152; 4 is 35153; 5 is 35154; 6 is 35155; 7 is 35156; 8 is 35157; 9 is 35158; 10 is 35159; 11 is 35160; 12 is 35161; 13 is 35162; 14 is 35163; 15 is 35164; 16 is 35165; 17 is... 35166; 18 is 35167; 19 is 35168; 20 is 35169; 21 is 35170; 22 is 35171; 23 is 35172; 24 is 35173; 25 is 35174; 26 is 35175; 27 is 35176; 28 is 35177; 29 is 35178; 30 is 35179; 31 is hesperidin plus TSB liquid medium.

[0027] Figure 6 A graph showing the difference in OD values ​​of bacteria in a TSB liquid culture medium with a hesperidin concentration of 0.4 mg / mL. Figure 6The numbers 1-31 represent the bacterial strain sequence; the total volume of the standard was adjusted to a concentration of 0.4 mg / mL, and the OD value represents the difference between the OD values ​​of the hesperidin mixed Sabouraud broth and the bacterial strain cultured for 48 h minus 0 h. 1 is 35201; 2 is 35202; 3 is 35203; 4 is 35204; 5 is 35205; 6 is 35206; 7 is 35207; 8 is 351208; 9 is 35209; 10 is 35210; 11 is 35211; 12 is 35212; 13 is 35213; 14 is 35214; 15 is 35215; 16 is 35216; 17 is 35216; 18 is 35216. 35217; 18 is 35218; 19 is 35219; 20 is 35220; 21 is 35221; 22 is 35222; 23 is 35223; 24 is 35224; 25 is 35225; 26 is 35226; 27 is 35227; 28 is 35228; 29 is 35229; 30 is 35330; 31 is hesperidin plus TSB liquid medium.

[0028] Figure 7 A graph showing the difference in OD values ​​of fungi in Sabouraud broth at a concentration of 0.4 mg / mL hesperidin. Figure 7 The values ​​1-31 represent the bacterial strain sequence; the total volume of the standard was adjusted to a concentration of 0.4 mg / mL, and the OD value represents the difference between the OD values ​​of the hesperidin-containing Sabouraud broth and the bacterial culture after 72 h minus 0 h. 1 is 19313; 2 is 19330; 3 is 19333; 4 is 19316; 5 is 19857; 6 is 19889; 7 is 19829; 8 is 19336; 9 is 19649; 10 is 19350; 11 is 19647; 12 is 19309; 13 is 19350; 14 is 19337; 15 is 19340; 16 is 19342; 17 is 19344; 18 is 19344; 19337 is 19340; 19342 is 19342; 19344 is 19344; 19337 is 19347; 19340 ...45 is 19344; 19345 is 19344; 19345 is 19345; 19351; 2019349; 21119352; 2219411; 2319419; 2419423; 2519425; 2619641; 2719817; 2819820; 2919827; 3019882; 31119885; 3219992; 33 is hesperidin plus Sabouraud broth.

[0029] Figure 8 The graph shows the difference in OD values ​​of fungi in carbon-free liquid culture medium with a hesperidin concentration of 0.1 mg / mL. Figure 8The numbers 1-31 represent the bacterial strain sequence; the total volume of the standard was adjusted to a concentration of 0.1 mg / mL, and the OD value represents the difference between the OD values ​​of the hesperidin mixed carbon-free liquid medium and the bacterial culture after 72 h minus 0 h. 1 is 19309; 2 is 19312; 3 is 19316; 4 is 19337; 5 is 19340; 6 is 19342; 7 is 19344; 8 is 19345; 9 is 19346; 10 is 19348; 11 is 19354; 12 is 19416; 13 is 19421; 14 is 19425; 15 is 19660; 16 is 19661; 17 is... 19662; 18 is 19816; 19 is 19817; 20 is 19820; 21 is 19823; 22 is 19825; 23 is 19827; 24 is 19849; 25 is 19851; 26 is 19853; 27 is 19882; 28 is 19883; 29 is 19888; 30 is 19992; 31 is hesperidin plus carbon-free liquid culture medium.

[0030] Figure 9 The graph shows the difference in OD values ​​of fungi in carbon-free liquid culture medium with a hesperidin concentration of 0.4 mg / mL. Figure 9 The numbers 1-31 represent the bacterial strain sequence; the total volume of the standard was adjusted to a concentration of 0.4 mg / mL. The OD values ​​represent the difference between the OD values ​​of the hesperidin mixed carbon-free liquid medium and the bacterial culture after 72 h minus 0 h. 1 is 19308; 2 is 19312; 3 is 19337; 4 is 19340; 5 is 19342; 6 is 19344; 7 is 19345; 8 is 19346; 9 is 19348; 10 is 19354; 11 is 194... 11; 12 is 19419; 13 is 19421; 14 is 19423; 15 is 19425; 16 is 19816; 17 is 19817; 18 is 19820; 19 is 19823; 20 is 19825; 21 is 19827; 22 is 19849; 23 is 19882; 24 is 19883; 25 is 19888; 26 is 19992; 27 is hesperidin plus carbon-free liquid culture medium.

[0031] Figure 10 The results were obtained from a carbon-free liquid culture medium with a hesperidin concentration of 0.4 mg / mL.

[0032] Figure 11 The results were obtained from a carbon-free liquid culture medium with a hesperidin concentration of 0.4 mg / mL.

[0033] Figure 12 The results were obtained from a carbon-free liquid culture medium with a hesperidin concentration of 0.4 mg / mL.

[0034] Figure 13 The results were obtained from a carbon-free liquid culture medium with a hesperidin concentration of 0.1 mg / mL.

[0035] Figure 14 The results were obtained from a carbon-free liquid culture medium with a hesperidin concentration of 0.1 mg / mL.

[0036] Figure 15 The value represents the OD difference of daidzein in the TSB medium in Example 3.

[0037] Figure 16 The value represents the OD difference of daidzein in Sabouraud medulloproteinase in Example 3.

[0038] Figure 17 The OD difference of daidzein under different concentrations of bacterial solutions in glucose-free culture medium in Example 3.

[0039] Figure 18 The results are obtained by high-performance liquid chromatography analysis of different strains in the 0.5 mg / mL daidzein glucose-free culture medium in Example 3.

[0040] Figure 19 The results are obtained by high-performance liquid chromatography analysis of different strains in the 0.1 mg / mL daidzein glucose-free culture medium in Example 3.

[0041] Figure 20 The results are obtained by high-performance liquid chromatography analysis of the 19425 transformant bacteria in the 0.1 mg / mL daidzein glucose-free culture medium in Example 3.

[0042] Figure 21 The results of high performance liquid chromatography (HPLC) analysis of transformant strain 19425 and daidzein in Example 3 are shown. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0044] The first aspect of this embodiment provides a method for screening flavonoid-transforming bacteria under carbon source stress, including the following steps:

[0045] Step 1: Prepare a carbon source stress screening medium, wherein the glucose concentration in the medium is 0-2.5 g / L, and the target flavonoid compound is added as the sole carbon source;

[0046] Step 2: After serially diluting the environmental samples, spread them onto the culture medium;

[0047] Step 3: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the difference in OD values ​​between 0 h of culture and after culture at a wavelength of 250-500 nm, and screen strains with an OD value difference ≥ 0.4 as candidate strains.

[0048] This invention provides a method for screening flavonoid-transforming bacteria under carbon source stress. By constructing a carbon source stress environment with a glucose concentration of 0-2.5 g / L and using the target flavonoid compound as the sole carbon source, the method induces reprogramming of microbial metabolic pathways, significantly improving the screening probability of highly efficient flavonoid-transforming strains. This method overcomes the limitations of traditional techniques, achieving a leapfrog improvement in screening efficiency—the carbon source stress mechanism drives the rapid enrichment of target microorganisms, shortens the screening cycle, increases the number of candidate strains, and, combined with ELISA quantitative detection and HPLC verification, reduces the false positive rate to below 5%. Simultaneously, it possesses significant industrialization value: simplified operation procedures reduce costs and improve detection accuracy.

[0049] To better understand the above technical solution, the following specific implementation methods are provided for further explanation.

[0050] Example 1

[0051] Screening of isoliquiritigenin-transforming bacteria using low-concentration carbon sources

[0052] Sample source: Soil samples from the Altun Mountains (JS018, JS013, ALJ031). The 66 fungal strains mainly belonged to 9 genera: *Aspergillus* sp., *Penicillium sensu stricto*, *Penicillium* sp., *Alternaria* sp., *Sarocladium* sp., *Serendipita* sp., *Chaetomium*, *Mortierella Coemans*, and *Bipolaris* sp.

[0053] Culture medium preparation:

[0054] PDA medium: 46.0 g of commercial potato dextrose agar medium, diluted to 1000 mL, and adjusted to pH 7.0.

[0055] TSB medium: 17.0 g tryptone, 3.0 g soybean peptone, 2.5 g dipotassium hydrogen phosphate, 5.0 g sodium chloride, 2.5 g glucose, 19.0 g agar powder, bring to a final volume of 1000 mL, adjust pH to 7.0.

[0056] Sabouraud dextrose medium: 10.0 g peptone, 40 g glucose, 19.0 g agar powder, bring to a final volume of 1000 mL, adjust to pH 7.0.

[0057] 10.0g LB tryptone, 5.0g yeast extract, 5.0g sodium chloride, 19.0g agar powder, bring to a final volume of 1000mL, adjust to pH 7.0.

[0058] Martin's medium: 2.5g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 5.0g peptone, 10.50g glucose, 19.0g agar powder, bring to a final volume of 1000mL, adjust pH to 7.0.

[0059] Licorice flavonoid culture medium: 10.0 g glycyrrhiza flavonoid extract, 0.5 g ammonium nitrate, 0.25 g dipotassium hydrogen phosphate, 0.125 g magnesium sulfate heptahydrate, bring to a final volume of 1000 mL, and adjust to pH 6.3-7.0.

[0060] Carbon source stress culture medium: 0.1 g isoliquiritigenin, 0.5 g ammonium nitrate, 0.25 g dipotassium hydrogen phosphate, 0.125 g magnesium sulfate heptahydrate, bring to a final volume of 1000 mL, and adjust to pH 6.3-7.0.

[0061] 1.4 Carbon medium: glucose 2.5 g / L, ammonium nitrate 2 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 1 g / L, pH 7 ± 0.5;

[0062] Sabouraud broth: 10.0g peptone, 40g glucose, 19.0g agar powder, bring to a final volume of 1000mL, and adjust to pH 7.

[0063] Filtering steps

[0064] (1) Activation of strain: Inoculate the strain into the culture medium and incubate at 37°C for 24 hours.

[0065] (2) Spread culture: Dilute-spread separation method: Weigh 1g of soil sample into a sterile PA bottle, then add 9mL of sterile water and mix thoroughly. Shake at 37℃ and 200r / min for 30min to promote the suspension of microorganisms and spores in the soil. Let stand at room temperature for 20min to allow large soil particles to settle. On a clean bench, use a pipette to draw 200μL of supernatant and transfer it to the surface of the solid culture medium. After sterilizing the spreader with an alcohol lamp flame, spread the bacterial solution evenly along the surface of the plate. Sterilize the edge of the culture dish with a flame, seal with sealing film after cooling, and incubate upside down in an incubator at 29-30℃ for 4 to 7 days.

[0066] Grinding method: In a cleanroom, place the pre-ground soil sample in a sterile empty petri dish. Wearing a sterile cap, take an appropriate amount of soil sample and spread it evenly on the surface of the culture medium. After the edge of the petri dish is briefly sterilized by flaming and cooled, seal it with sealing film and incubate it upside down in an incubator at 29-30℃ for 4 to 6 days.

[0067] (3) Initial screening: Selective isolation of fungi: After the solid culture medium has cooled to about 60°C, add antibiotics at a ratio of 1000:1 (e.g., 250 μL to 250 mL of culture medium), mix well, and pour into culture dishes, avoiding bacterial coverage to promote fungal growth. Briefly sterilize the culture dish lids by flaming them over an alcohol lamp flame. After the dishes have cooled, seal them with plastic wrap and invert them in a 30°C incubator for 4 to 7 days.

[0068] (4) Secondary screening: Streak plate method: This method is suitable for purifying bacteria and actinomycetes. Use a sterile bamboo stick or inoculation loop to pick up single colonies of different colors and morphologies, and draw uniform and continuous straight lines on the surface of the solid culture medium using the three-zone streak method. To ensure that single colonies are formed in the final area, the bamboo stick needs to be sterilized by flame after each zone streak. Keep the pressure even and moderate when strewing to avoid breaking the culture medium. Briefly flame the edge of the petri dish for sterilization, and after cooling, seal it with plastic wrap and invert it in a 30℃ incubator for 1-2 days.

[0069] Inverted pipette tip isolation method: This method is suitable for purifying large fungi or fungi whose spores are easily dispersed, avoiding the problems of spore dispersion and contamination, and the inability to grow single colonies, that occur with the traditional streak method. Take a sterile pipette tip (200 μL), with the tip pointing upwards, and gently invert the tip onto the target colony. Slightly open the petri dish lid and gently flick the pipette tip with your finger to allow the fungal cells or spores to fall into the center of the culture medium. Briefly flank the edge of the petri dish under an alcohol lamp flame, and after cooling, seal it with plastic wrap and incubate it upside down in a 30°C incubator for 2-3 days. Then, identify the strain.

[0070] (5) Stress culture: Inoculate the bacterial solution into the carbon source stress culture medium at an inoculation rate of 10% and culture at 30°C with shaking for 48 hours.

[0071] (6) Microplate reader detection: Further, a 1 mg / mL isoglycyrrhizin standard was prepared, with a total standard concentration of 100 μg / mL in the mixture. Three experimental groups were set up. In the experimental group, 675 μL of Sabouraud broth and 150 μL of isoglycyrrhizin standard were added to a 2 mL sterile centrifuge tube, and the volume was adjusted to 1.5 mL by pipetting the 675 μL of pre-shaken bacterial culture. In the bacterial culture control group, no standard was added, only bacterial culture and culture medium were added to adjust the volume to 1.5 mL. In the standard control group, only 1350 μL of Sabouraud broth and 150 μL of standard were added. 200 μL of sample from each of the three groups was added to a 96-well plate, and the OD value changes at wavelengths of 250-500 nm within 0 h were detected using a full-wavelength microplate reader. The wavelengths at 366nm for fungal detection samples within 0h, 72h, and 7d, and the wavelengths at 366nm for bacterial detection samples within 0h, 24h, and 72h, were used to screen strains with OD values ​​greater than ±0.4 within 0-72h.

[0072] (7) HPLC verification: Sample preparation: Take 500 mL of the sample to be tested into a 1.5 mL centrifuge tube and centrifuge at 12000 r / min for 10 min. Take the supernatant, add an equal volume of methanol, filter through a 0.45 μm filter membrane into a liquid chromatography vial, and store it in a refrigerator at 4℃ for later use.

[0073] Prepare the mobile phase according to the following ratio: Phase A (aqueous phase) 1% formic acid solution, Phase B (organic phase) acetonitrile:methanol = 9:1. Mix the mobile phase with a magnetic stirrer (20 min), filter, and sonicate for 5 min to avoid bubble interference. Set the flow rate (1 mL / min), column temperature (35℃), and detection wavelength. Perform automatic venting of the mobile phase, create a batch processing file, and ensure the accuracy of the pressure, flow rate, chromatographic temperature, and method file before starting the detection. After the experiment, rinse the column with pure methanol for 30 min to avoid salting out or impurity residue. Export and save the experimental data, and use Origin Pro 2024 software to plot the curves of the test sample at 370 nm and 280 nm, calculating its retention time and peak area.

[0074] Test results are as follows Figure 1-4 As shown. Figure 1 JS018 soil sample was prepared using the grinding method. Figure 1 The strains isolated by f) in the dilution plating method were higher than those isolated by the dilution plating method. Figure 1 e) has a great advantage in terms of the richness of bacterial species. Using TSB and LB media, a richer variety of bacteria and actinomycetes can be isolated from soil samples from the Altyn Mountains.

[0075] Figure 2 To determine the purification results of the strain, after co-culturing the ISL standard with the bacterial culture for a period of time, if the strain has the ability to transform the standard, its absorbance value will change. Methodological validation showed that the peak value of ISL is at 366 nm. Therefore, by calculating the change in absorbance value (OD difference) at 366 nm within 0-72 hours, strains with an OD difference of ±0.4 can be efficiently screened.

[0076] Figure 3 As shown, the absorbance values ​​of TSB medium and Sabouraud dextrose culture at 250-500 nm changed very little within 0-72 h, ruling out the influence of the medium on the standard. Therefore, strains with large absorbance value changes within 0-72 h can be considered as isoglycyrrhizin-transforming bacteria. A total of 17 strains had OD differences exceeding ±0.4, namely: 18903, 18904, 18902, 18908, 18917, 18915, 18920, 18930, 18932, 18935, 18935, 18933, 18936, 18939, 18940, 18955, and 18957.

[0077] High-performance liquid chromatography (HPLC) analysis revealed that the peak values ​​of ISL were at 370 nm and 280 nm. Since the test sample contained an ISL standard, it also exhibited similar peak values ​​at 370 nm and 280 nm. If the bacterial strain in the test sample has the ability to transform isoglycyrrhizin, several scenarios may occur: first, the peak area of ​​the test sample will be lower than that of the pure standard; second, a new peak will appear near the peak value of the standard; and third, the peak retention time of the experimental group will shift compared to the control group. This experiment used Sabouraud dextrose agar and carbon source stress medium, combined with UV induction technology, to conduct related research. The results are as follows... Figure 4 As shown.

[0078] The results are as follows Figure 4 As shown, due to the low purity of the ISL standard (≥98%) and the presence of 4% glycyrrhizin, two peaks appeared at 280 nm. Mass spectrometry analysis revealed that glycyrrhizin peaked around 14 min, while isoglycyrrhizin peaked around 17 min. Compared to the control group, the peak values ​​in the experimental group were significantly lower, indicating that these bacteria possessed the ability to absorb and consume the isoglycyrrhizin standard.

[0079] In a mixed culture experiment on a carbon source stress medium, one fungus was found to inhibit the growth of the other strains, indicating that the strain may have antibacterial activity.

[0080] Seven strains potentially capable of isoglycyrrhizin conversion were screened using an enzyme-linked immunosorbent assay (ELISA) reader. The peak values ​​of these seven strains were significantly reduced in the high-performance liquid chromatography (HPLC) chromatograms, indicating their ability to utilize and consume ISL. Furthermore, the peak values ​​of strains 18936, 18903, and 18940 showed slight shifts, possibly due to ISL glycosylation. Because the standard sample had low purity (≥98%) and contained 4% glycyrrhizin, two distinct peaks appeared at 280 nm. The peak area of ​​isoglycyrrhizin in the control group (carbon source stress medium + standard sample) was significantly lower than that of the pure standard, while the peak value of glycyrrhizin was relatively higher. This may be because ISL and glycyrrhizin are isomers, and their unstable chemical structures lead to natural conversion to glycyrrhizin in the carbon source stress medium. After co-culturing strain 18933 (Alternaria dauci) with the standard sample in the carbon source stress medium for 14 days, the peak value of isoglycyrrhizin (ISL) was significantly higher than that of the control group and the pure standard. This phenomenon is highly consistent with the results reported by Guo et al. on improving the purity of baicalein through microbial transformation, confirming that strain 18933 Alternaria dauci may have used impurities in the standard sample to synthesize ISL with higher purity. This hypothesis has high rationality and credibility, indicating that the strain may have the ability to synthesize isoglycyrrhizin, which is expected to be used to improve the purity and yield of ISL.

[0081] Example 2

[0082] Gradient carbon source screening for hesperidin-transforming bacteria

[0083] Sample source: 100 bacterial strains (Bacillus, Pseudomonas, etc.) isolated from soil around the Tashkurgan River and Kizilsu River.

[0084] Culture medium preparation:

[0085] Carbon-free liquid culture medium: 1g dimethyl bicarbonate, 2g ammonium nitrate, 0.5g anhydrous magnesium sulfate. Note: The high-pressure steam temperature is 115℃.

[0086] TSB liquid medium: 17.0g tryptone, 3.0g soybean peptone, 5.0g sodium chloride, 2.5g dipotassium hydrogen phosphate, 2.5g glucose.

[0087] Sabouraud dextrose: 40.0g, peptone 10.0g, pH (approx. 5.6, no adjustment required).

[0088] Filtering steps

[0089] (1) Strain activation: The glycerol culture tubes were removed from the -80℃ ultra-low temperature freezer and thawed at room temperature (25±2℃). Simultaneously, TSB liquid medium (17g / L tryptone, 3g / L soybean peptone, 5g / L sodium chloride, 2.5g / L dipotassium hydrogen phosphate, 2.5g / L glucose) and Sabouraud broth (40g / L glucose, 10g / L peptone), sterilized by autoclaving at 121℃ for 20 min, were prepared. 3–5 mL of glycerol culture was transferred to 10 mL sterile centrifuge tubes using aseptic techniques. Appropriate culture parameters were set according to the strain characteristics (example parameters: aerobic bacteria were cultured in a 37℃ constant temperature incubator with shaking at 134 rpm for 12–24 h). Cell proliferation monitoring: Turbidity analysis was used to monitor cell growth; significant turbidity changes were observed after 12–24 h of routine culture. Activated cells were wrapped in plastic wrap and stored in a clean environment at 4℃ for short-term preservation.

[0090] (2) Spread culture: Dilute-spread separation method: Weigh 1g of soil sample into a sterile PA bottle, then add 9mL of sterile water and mix thoroughly. Shake at 37℃ and 200r / min for 30min to promote the suspension of microorganisms and spores in the soil. Let stand at room temperature for 20min to allow large soil particles to settle. On a clean bench, use a pipette to draw 200μL of supernatant and transfer it to the surface of the solid culture medium. After sterilizing the spreader with an alcohol lamp flame, spread the bacterial solution evenly along the surface of the plate. Sterilize the edge of the culture dish with a flame, seal with sealing film after cooling, and incubate upside down in an incubator at 29-30℃ for 4 to 7 days.

[0091] Grinding method: In a cleanroom, place the pre-ground soil sample in a sterile empty petri dish. Wearing a sterile cap, take an appropriate amount of soil sample and spread it evenly on the surface of the culture medium. After the edge of the petri dish is briefly sterilized by flaming and cooled, seal it with sealing film and incubate it upside down in an incubator at 29-30℃ for 4 to 6 days.

[0092] (3) Initial screening: The strains were inoculated into the culture medium and cultured at 30°C for 72 hours.

[0093] (4) Secondary screening: Streak plating method: This method is suitable for purifying bacteria and actinomycetes. Use a sterile bamboo stick or inoculation loop to pick up single colonies with different colors and morphologies, streak them on the surface of a solid culture medium using the three-zone method, seal them with plastic wrap, and incubate them upside down in a 30℃ incubator for 1-2 days.

[0094] Inverted pipette tip isolation method: This method is suitable for purifying large fungi or fungi whose spores are easily dispersed, avoiding the problems of spore dispersion and contamination, and the inability to grow single colonies, that occur with the traditional streak method. Take a sterile pipette tip (200 μL), with the tip pointing upwards, and gently invert the tip onto the target colony. Slightly open the petri dish lid and gently flick the pipette tip with your finger to allow the fungal cells or spores to fall into the center of the culture medium. Briefly flank the edge of the petri dish under an alcohol lamp flame, and after cooling, seal it with plastic wrap and incubate it upside down in a 30°C incubator for 2-3 days. Then, identify the strain.

[0095] (5) Stress culture: Inoculate the bacterial solution into the carbon source stress culture medium at an inoculation rate of 10% and culture at 30°C with shaking for 48 hours.

[0096] (6) ELISA reader detection: For the construction of experimental transformation groups, the strains activated for 24 hours on Sabouraud agar (fungi) or TSB agar (bacteria) were mixed with hesperidin solution (1 mg / mL) and its corresponding liquid culture medium in a strict ratio in a sterilized 2 mL centrifuge tube.

[0097] Establish experimental transformation groups of two concentrations of hesperidin standard.

[0098] Bacteria: Experimental procedure for the 0.4 mg / mL hesperidin standard concentration experimental group: Use a pipette to take hesperidin solution (1 mg / mL), bacterial suspension of the strain to be transformed, and liquid culture medium in a certain proportion into 2 mL centrifuge tubes.

[0099] Fungi: Experimental procedure for the 0.4 mg / mL hesperidin standard concentration experimental group: Use a pipette to take hesperidin solution (1 mg / mL), bacterial culture of the strain to be transformed, and Sabouraud liquid medium and add them to a 2 mL centrifuge tube at a certain ratio.

[0100] Fungi: Experimental procedure for the 0.1 mg / mL hesperidin standard concentration experimental group: Use a pipette to take hesperidin solution (1 mg / mL), bacterial culture of the strain to be transformed, and carbon-free liquid culture medium in a certain proportion and add them to a 2 mL centrifuge tube.

[0101] Fungi: Experimental procedure for the 0.4 mg / mL hesperidin standard concentration experimental group: Use a pipette to take hesperidin solution (1 mg / mL), bacterial culture of the strain to be transformed, and carbon-free liquid culture medium in a certain proportion and add them to a 2 mL centrifuge tube.

[0102] The reason for establishing experimental groups with standard concentrations of 0.4 mg / mL and 0.1 mg / mL hesperidin is that hesperidin has antibacterial properties at high concentrations, so the concentration of hesperidin during transformation is reduced. Two concentration groups are used in the carbon-free culture system because there is no carbon source in the culture system, and hesperidin is the only way to provide carbon for fungi. A concentration of 0.1 mg / mL may result in insufficient carbon source, preventing normal growth, reproduction, and transformation of the fungi. Therefore, the concentration of hesperidin is increased to 0.4 mg / mL.

[0103] In the preliminary experimental stage, a full-wavelength microplate reader was used for spectral scanning (250–500 nm), and the characteristic absorption spectrum of hesperidin was determined through three repeated scans. Validation with a blank control group showed a significant absorption peak at 283 nm. This wavelength detection strategy, after methodological validation, can effectively shorten the detection time in the transformation bacteria screening process and improve the efficiency of subsequent data processing.

[0104] The transformation culture cycle was set according to microbial growth kinetics; the bacterial experimental group had three time points: 0, 24, and 48 hours (37℃, 134 rpm), while the fungal experimental group had three time points: 0, 72, and 168 hours (28℃, 134 rpm). Full-wavelength scanning and single-wavelength point detection were performed at each time point. The 0-hour full-wavelength data served as a baseline reference for subsequent qualitative analysis of metabolite absorption peak shifts and newly added characteristic peaks. The time point intervals were set with reference to previous growth curve experiments to ensure coverage of the entire process from the logarithmic growth phase to the stationary phase.

[0105] (7) HPLC validation: Column: ZORBAX Eclipse Plus C18 (250nm×4.6nm, 5μm); gradient elution program: 0–5 min, 10% B; 5–8 min, 15% B; 8–10 min, 50% B; 10–18 min, 70% B; 18–28 min, 85% B; 28–32 min, 100% B; 32–40 min, 5% B. Mobile phase: 0.1% formic acid water (solvent A) and acetonitrile:methanol = 9:1 (solvent B), flow rate: 1.0 mL / min, injection volume: 10 μL, detection wavelength: 280 nm.

[0106] Test results are as follows Figure 5-14 As shown.

[0107] The results of microbial transformation of hesperidin were screened using an enzyme-linked immunosorbent assay (ELISA) reader, such as Figure 5 Bacterial strains 35163 and 35164, cultured in a 0.4 mg / mL hesperidin-mixed TSB liquid medium, showed a significant difference in OD values ​​at 283 nm compared to the control group (hesperidin plus TSB liquid medium), suggesting possible transformation. Figure 6 Bacterial strains 35204, 25211, and 35219, cultured in a 0.4 mg / mL hesperidin-mixed TSB liquid medium, showed significantly different OD values ​​at 283 nm compared to the control group, suggesting possible transformation. Figure 7 Bacterial strains 19309 and 19337, cultured in a mixture of hesperidin and Sabouraud broth at a concentration of 0.4 mg / mL, showed significantly different OD values ​​at 283 nm compared to the control group (hesperidin plus Sabouraud broth), suggesting potential transformation. Figure 8 The bacterial strain 19425, cultured in a carbon-free liquid medium with a hesperidin concentration of 0.1 mg / mL, showed a significantly different OD value at 283 nm compared to the control group (hesperidin plus carbon-free liquid medium), suggesting potential transformation. Figure 9 The bacterial strains 19308, 19344, 19423, and 19827, which were cultured in a carbon-free liquid medium with a concentration of 0.4 mg / mL hesperidin, showed a significant difference in OD values ​​at 283 nm compared to the control group (hesperidin plus carbon-free liquid medium), suggesting possible transformation.

[0108] Strains potentially capable of transformation were screened using a full-wavelength microplate reader combined with thin-layer chromatography. These strains were then filtered through a sterile 0.22 μm microporous membrane into liquid chromatography vials and detected by high-performance liquid chromatography (HPLC). Figure 10-13 It was found that the peak area of ​​the standard was reduced at a wavelength of 280 nm for some strains. Subsequent experiments proved that the reason for the decrease in the peak area was the presence of air bubbles in the liquid during the passage of the mobile phase through the liquid chromatography column, causing fluctuations in the peak area. This was not due to the transformation of hesperidin by the strains. Figure 14 It can be seen that strain 19425 of Aspergillus niger showed a new peak compared with the hesperidin standard at a retention time of 19-20 min, indicating the presence of transformed substances.

[0109] This study used a full-wavelength ELISA reader combined with high-performance liquid chromatography (HPLC) to comprehensively examine the ability of 100 bacterial strains to transform hesperidin. The results showed that, under the existing conditions, one strain among all strains possessed hesperidin-transforming activity. The full-wavelength ELISA method, with its advantages of high throughput and rapid detection, effectively narrowed down the initial screening range; HPLC ultimately quantitatively confirmed the reliability of the transforming strains through changes in characteristic peak areas.

[0110] Through screening of these 100 bacterial strains to transform hesperidin, it was found that the bacterial transformation ability of hesperidin was not as strong as that of fungi, and the carbon source stress medium had the best effect among the three culture media.

[0111] Example 3

[0112] Screening of soybean aglycone transforming bacteria for carbon source stress

[0113] Sample source: Endophytic fungi from Ural licorice leaves (37 strains of fungi, including strain 19425). A preliminary selection included 43 bacteria and 37 fungi. (Fungal strains included: *Aspergillus fumigatus*, *Penicillium*, etc.; bacterial strains included: *Microbranchium*, *Streptomyces*).

[0114] Culture medium preparation:

[0115] TSB medium: 8.5g tryptone, 1.5g soybean peptone, 1.25g dipotassium hydrogen phosphate, 2.5g sodium chloride, 1.25g glucose, bring to a final volume of 500mL, and adjust pH to 7.0.

[0116] LB medium: 5.0 g tryptone, 2.5 g yeast extract, 2.5 g sodium chloride, 9.0 g agar powder, bring to a final volume of 500 mL, and adjust pH to 7.0.

[0117] Carbon source stress culture medium: ammonium nitrate 1g, magnesium sulfate 0.25g, dipotassium hydrogen phosphate 0.5g, bring to a final volume of 500mL, pH 7.

[0118] Filtering steps

[0119] (1) Strain activation: Based on the strain information obtained from the strain library, fungi (such as 19827, 19425, etc.) preserved in glycerol tubes were activated as seed culture in 10mL centrifuge tubes or PA bottles containing Sabouraud broth. For bacteria (such as 50100, 50280, etc.), activation was carried out in an environment containing TSB broth. Subsequently, the centrifuge tubes or PA bottles containing the strains were placed in a constant temperature shaker for 24 hours of incubation. During the incubation period, the growth of the colonies needed to be closely monitored to ensure that the activity of the strains was fully restored.

[0120] (2) Spread culture: Based on the strain information obtained from the strain library, fungi (such as 19827, 19425, etc.) preserved in glycerol tubes were activated as seed culture in 10mL centrifuge tubes or PA bottles containing Sabouraud broth. Bacteria (such as 50100, 50280, etc.) were activated in an environment containing TSB broth. Subsequently, the centrifuge tubes or PA bottles containing the strains were placed in a constant-temperature shaker for 24 hours of incubation. During the incubation period, the growth of the colonies needed to be closely monitored to ensure that the strain activity was fully restored.

[0121] (3) Initial screening: The strains were inoculated into the culture medium and cultured at 30°C for 72 hours.

[0122] (4) Second screening:

[0123] Fungal transformation experiment

[0124] After activation, the fungal culture was aspirated, and mycelia were picked. Then, Sabouraud broth was aspirated, and finally, 1 mg / mL daidzein solution was added to bring the daidzein concentration to 0.5 mg / mL. Following the same experimental procedure, Sabouraud broth was replaced with glucose-free broth to achieve daidzein concentrations of 0.1 mg / mL and 0.5 mg / mL. The culture was placed in a constant-temperature shaker at 37°C and 200 rpm for 48 h, 72 h, and 168 h, during which colony morphology and color changes were observed regularly.

[0125] Bacterial transformation experiment

[0126] The activated bacterial strain was aspirated and added to TSB culture medium and 1 mg / mL daidzein solution to achieve a concentration of 0.3 mg / mL. The mixture was then placed in a constant-temperature shaker and incubated at 37°C and 200 rpm for 24 h and 48 h, with colony changes observed and recorded every 24 hours.

[0127] (5) Stress culture: Inoculate the bacterial solution into the carbon source stress culture medium at an inoculation rate of 10% and culture at 30°C with shaking for 48 hours.

[0128] (6) ELISA reader detection:

[0129] Specificity: Add 200 μL of methanol, daidzein solution, TSB medium, Sabouraud broth, or glucose-free medium to the enzyme-labeled strip, and detect the OD value using an enzyme-labeled reader.

[0130] Linearity: Dissolve 1 mg of daidzein in a 10 mL volumetric flask, dilute to volume with methanol, and shake well to obtain a 100 μg / mL stock solution of the reference standard. Add the above reference standard from 200 μL to the enzyme-linked immunosorbent assay (ELISA) strip using the differential dilution method, adding methanol solution to each well to a final volume of 200 μL. Measure the OD values ​​at wavelengths of 200–650 nm using an ELISA reader. Dilute the reference standard 20-fold and 100-fold and measure their OD values ​​using the same method.

[0131] In the experiment, two groups were set up for each strain: a blank control group and an experimental group. In the blank control group, vanillin sulfate, culture medium, and bacterial suspension were added to each well and mixed well. In the experimental group, vanillin sulfate, culture medium, and daidzein-transforming bacterial suspension were added to each well. The wavelength was selected as 200-650 nm. The absorbance values ​​at different time points (0h, 72h, and 168h for fungi; 0h, 24h, and 48h for bacteria) were measured. The difference between the termination time (168h or 48h) and the start time (0h) of the daidzein standard bacterial suspension added at a wavelength of 300 nm was calculated to screen out values ​​with larger differences. The data showed that values ​​with larger differences may indicate transformation efficiency. According to the reference, the absorbance of fungi increased significantly at 72h, showing a higher transformation efficiency; while the absorbance of bacteria tended to stabilize after reaching a peak at 48h. By comparing the changes in absorbance, the optimal transformation time points for fungi and bacteria could be determined, providing a scientific basis for subsequent large-scale culture and product extraction.

[0132] (7) HPLC Validation: HPLC typically uses a reversed-phase column, such as a C18 column, as the stationary phase and a polar solvent, such as formic acid-water or acetonitrile-methanol, as the mobile phase. Daidzein has moderate polarity, and the difference in the partitioning of hydroxyl and ketone groups between the stationary and mobile phases determines its retention time. Driven by a high-pressure pump, the mobile phase passes through the column, and daidzein gradually separates from impurities due to their polarity differences, forming independent chromatographic peaks.

[0133] The bacterial culture containing daidzein was centrifuged at 12,000 rpm for 10 min. The supernatant was then diluted with an equal volume of methanol solution and mixed thoroughly. The mixture was filtered through a 0.45 μm filter membrane, and the filtrate was collected for high-performance liquid chromatography (HPLC) analysis. A C18 column was used. Mobile phase A was formic acid-water (1:99 v / v), and mobile phase B was acetonitrile-methanol (9:1 v / v). The flow rate was set at 1.0 mL / min, and the column temperature was maintained at 30 °C. Daidzein and its transformation products in the filtrate were separated and detected under a UV detector at a wavelength of 280 nm. By comparing the chromatograms of the standard bacterial culture and the sample, the presence of daidzein and its transformation products in the sample could be qualitatively analyzed. Simultaneously, quantitative analysis was performed based on peak area to further evaluate the transformation efficiency.

[0134] When using an ELISA reader for analysis, data from bacterial strains at 0h and 48h, and fungal strains at 0h, 72h, and 168h, were selected for analysis. Figure 15-17 As shown, the OD difference of bacterial cultures cultured on glucose-free medium and Sabouraud broth at different time points was measured using an ELISA reader. Data calculation and analysis were then performed, yielding the following results. Using the OD difference of the standard sample as a benchmark, bacterial cultures with OD differences greater than those of the standard sample were selected.

[0135] The comparison is based on the OD difference value being greater than that of the standard product, and where the two OD differences differ significantly. Figure 15-16 Analysis revealed that among bacterial strains, the OD difference between the strains and the standard was not significantly different. Among fungal strains, strains 19116, 19117, 19309, 19335, 19340, 19344, 19425, 19816, 19823, and 19889 showed larger differences in 72h OD and 168h OD, while strain 19425 showed a relatively larger difference at 168h OD, suggesting that the standard might be utilized by the strains to generate other substances. To verify this hypothesis, the transformed bacteria were subjected to further thin-layer chromatography analysis to further screen whether these fungal strains possessed the ability to transform new substances.

[0136] After performing high performance liquid chromatography analysis, such as Figure 18 and 19 As shown, the strain was more likely to transform at a daidzein concentration of 0.1 mg / mL than at 0.5 mg / mL. Figure 20 The results show that new peaks appeared at 5 min and 20 min, proving that the 19425 bacterial culture utilized daidzein for conversion. The converted substance dissolved in the mobile phases at 5 min and 20 min. Mobile phase A was formic acid:water = 1:99, and mobile phase B was methanol:acetonitrile = 1:9. The concentration of mobile phase B was low at 5 min and high at 20 min. Figure 20 As shown, the high-performance liquid chromatography (HPLC) analysis results of 19425, compared with daidzein culture medium plus carbon-free medium and methanol, prove that the peak value indicates the appearance of a new substance, suggesting that the extracellular enzyme produced by 19425 bacteria consumed daidzein to generate a new substance. Figure 20 Analysis shows that the substance appearing at 5 minutes is soluble in water, while the substance appearing at 20 minutes is soluble in organic solvents.

[0137] Depend on Figure 21The addition of daidzein to strain 19425 of Aspergillus niger and its comparison with the daidzein solution revealed a small peak at around 4 minutes and three peaks at around 16 minutes. This demonstrates that daidzein was converted into a new substance by enzymes in the Aspergillus niger bacterial solution, and the substance appearing at 16 minutes may have a structure similar to daidzein.

[0138] The 16rsDNA of Aspergillus niger and Alternaria carota are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0139] This experiment compared the conversion efficiency of different culture media. The fungal conversion rate of sugar-free medium was much higher than that of Sabouraud medium, indicating that the carbon source may limit the strain's ability to convert daidzein.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for screening flavonoid-transforming bacteria under carbon source stress, characterized in that, Includes the following steps: Step 1: Prepare a carbon source stress screening medium, wherein the glucose concentration in the medium is 0-2.5 g / L, and the target flavonoid compound is added as the sole carbon source; Step 2: After serially diluting the environmental samples, spread them onto the culture medium; Step 3: Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the difference in OD values ​​between 0 h of culture and after culture at a wavelength of 250-500 nm, and screen strains with an OD value difference ≥ 0.4 as candidate strains.

2. The method according to claim 1, characterized in that, The carbon source stress culture medium includes ammonium nitrate 0.5-2 g / L; dipotassium hydrogen phosphate 0.25-1 g / L; magnesium sulfate 0.125-0.5 g / L; flavonoids 0.1-0.5 g / L; pH 6.3-7.

0.

3. The method according to claim 2, characterized in that, The flavonoid compound is selected from isoliquiritin, hesperidin, or daidzein.

4. The method according to claim 3, characterized in that, The OD value detection wavelengths are as follows: 366nm for isoliquiritin detection, 283nm for hesperidin detection, and 280-300nm for daidzein detection.

5. The method for screening flavonoid-transforming bacteria under carbon source stress according to claim 1, characterized in that, In step 2, incubate at 25-35℃ for 3-7 days.

6. The method according to claim 1, characterized in that, The strain screening criteria in step 3 include: fungi: OD difference ≥ 0.4 within 0-72h of culture; bacteria: OD difference ≥ 0.4 within 0-48h of culture.

7. The method according to any one of claims 1-6, characterized in that, The experiment also includes step 4, which verifies the strain's ability to convert flavonoids by HPLC, with a positive standard being a new product peak or a peak area change of ≥20%.

8. The method according to claim 7, characterized in that, The HPLC validation conditions were as follows: C18 column, mobile phase A was 1% formic acid water, mobile phase B was acetonitrile:methanol = 9:1, flow rate was 1.0 mL / min, column temperature was 30-35℃, and detection wavelength was 280 nm or 370 nm.

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