MTHM-based method for rapidly screening isoliquiritigenin transformed strains in large scale

Abstract

The invention relates to the technical field of strain screening, in particular to a method for rapidly screening isoliquiritigenin transformed strains on a large scale on the basis of MTHM, a microplate reader is adopted for rapid screening, a transformed product is verified through thin-layer chromatography, high performance liquid chromatography and liquid mass spectrometry, the detection precision is amplified step by step, and the isoliquiritigenin transformed strains can be obtained. The screening efficiency of the isoliquiritigenin transforming bacteria is obviously improved. The method not only is efficient and reliable, but also provides a new technical thought and theoretical basis for development of natural products and application of microbial conversion.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of strain screening, and particularly relates to a method for rapidly and large-scale screening of isoliquiritigenin conversion strains based on MTHM. BACKGROUND

[0002] The roots and rhizomes of Glycyrrhiza uralensis are rich in flavonoids, which are widely used in traditional Chinese medicine and natural additives. Flavonoids are important polyphenols in plant medicines, and have various physiological functions such as anti-inflammatory, antioxidant, antitumor, and antibacterial. Glycyrrhiza contains more than 300 flavonoids, among which chalcones are the most important secondary metabolites and are precursors of flavonoids, and have various biological activities. Isoliquiritigenin is one of the main active flavonoids in Glycyrrhiza, and has three phenolic hydroxyl groups in its molecular structure, which limits its transmembrane transport capacity. However, the rich substitution sites on the benzene ring provide conditions for structural modification, and it is expected to enhance its biological activity through structural modification. Previous studies have improved the solubility and stability of isoliquiritigenin through structural modification, or enhanced its antitumor effect by introducing different groups.

[0003] Most studies on isoflavones and flavonoids focus on plant sources, while microbial sources are less studied. However, it has been confirmed that there is a biosynthetic metabolic pathway of isoflavones and flavonoids in microorganisms, especially in fungi. Many important drugs are derived from secondary metabolites of fungi, and microbial biotransformation has the potential to produce new bioactive flavonoids. In recent years, microbial biotransformation has attracted attention as a cost-effective and environmentally friendly tool for drug design. Microorganisms can convert chalcones into various new flavonoids through biotransformation processes such as cyclization, hydroxylation, reduction, demethylation, and dehydrogenation. For example, different strains can perform hydroxylation, demethylation, cyclization, or hydrogenation reactions on chalcones to generate flavonoids with different structures. Cunninghamella, Penicillium, and Aspergillus strains have strong conversion ability for flavonoids and can perform various reactions.

[0004] Therefore, it is of great importance to develop a rapid and efficient large-scale method for screening isoliquiritigenin conversion strains. SUMMARY

[0005] The present application aims at the problems existing in the prior art, and provides a method for rapidly screening isoliquiritigenin conversion strains on a large scale based on MTHM. The conversion product is verified by using an enzyme marker instrument for rapid screening, thin layer chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry, and the detection precision is amplified step by step, so that the screening efficiency of the isoliquiritigenin conversion strain is significantly improved. The method is not only efficient and reliable, but also provides a new technical idea and theoretical basis for the development of natural products and the application of microbial conversion.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] A method for rapidly screening isoliquiritigenin conversion strains on a large scale based on MTHM, comprising the following steps:

[0008] Step 1, respectively mix the to-be-screened bacterial liquid and the isoliquiritigenin solution to obtain a first mixed liquid; detect the absorbance value of the first mixed liquid by using an enzyme marker instrument; culture the first mixed liquid for more than 48 hours to obtain a second mixed liquid, and detect the absorbance value of the second mixed liquid by using an enzyme marker instrument; the absolute value of the difference between the two absorbance values is greater than 0.4 as the preliminary screened strain;

[0009] Step 2, respectively detect the mixed solution of the preliminary screened strain and the isoliquiritigenin solution, and the isoliquiritigenin standard by using thin layer chromatography; select the strain with different development positions of the mixed solution and the isoliquiritigenin standard and two or more development positions of the mixed solution as the second screened strain;

[0010] Step 3, respectively detect the mixed liquid of the second screened strain and the isoliquiritigenin solution by HPLC, and observe the appearance of other peaks on the side of the peak position of the isoliquiritigenin standard or the change of the peak type of the mixed liquid and the peak type of the standard on the chromatogram, then the strain is regarded as the third screened strain;

[0011] Step 4, respectively mix the bacterial liquid of the third screened strain and the isoliquiritigenin solution and culture for more than 48 hours, and detect the conversion product of the strain under the ultra-high performance liquid chromatography-mass spectrometry to screen the final isoliquiritigenin conversion strain.

[0012] The present application provides a method for rapidly screening isoliquiritigenin conversion strains on a large scale based on MTHM, which adopts enzyme marker instrument for rapid screening, thin layer chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry to verify the conversion product, and significantly improves the screening efficiency of the isoliquiritigenin conversion strain by amplifying the detection precision step by step. The method is not only efficient and reliable, but also provides a new technical idea and theoretical basis for the development of natural products and the application of microbial conversion.

[0013] Further, in step 1, the to-be-screened bacterial liquid is obtained by strain activation treatment:

[0014] When the strain is bacteria, the strain is inoculated into TSB medium and cultured at 36-38°C in an incubator for 1-3 days.

[0015] When the strain is fungi, the strain is inoculated into Czapek medium and cultured at 26-30°C in an incubator for 5-7 days.

[0016] Further, in step 1, the first mixed solution is cultured for 48 hours.

[0017] Further, in step 1, the optimal concentration of the enzyme label instrument detection of isoliquiritigenin standard solution is 5-100 μg / mL.

[0018] The optimal concentration of the enzyme label instrument detection of isoliquiritigenin standard solution is 5-100 μg / mL. If the concentration is too high or too low, the isoliquiritigenin standard solution does not show a good linear relationship. The OD value of the mixed solution of the 48-hour culture and the isoliquiritigenin solution is subtracted from the 0-hour OD value, and the absolute value of the OD difference greater than 0.4 is used as the preliminary screening of the strain for the next experiment.

[0019] Further, in step 2, in the thin layer chromatography operation, the developing agent includes the following raw materials in a volume ratio: methanol: ethyl acetate: petroleum ether = 1:10:16. Research has found that controlling the addition amount of each raw material in the developing agent within a reasonable range can better control the development degree, thereby more efficiently screening the strain.

[0020] Further, in step 3, the mobile phase for HPLC detection is acetonitrile: methanol (9:1)-1% formic acid water. Reasonable control of the mobile phase can ensure the appearance of the peak type, thereby more efficiently screening the strain.

[0021] Further, in step 4, under the detection of ultra-high performance liquid chromatography-mass spectrometry, the mobile phase A is water, the mobile phase B is methanol, and the injection amount is 10 μL.

[0022] In summary, due to the adoption of the above technical solutions, the present application has the following advantages:

[0023] The present application provides a method for rapidly screening isoliquiritigenin conversion strains on the basis of MTHM, which adopts enzyme label instrument rapid screening, thin layer chromatography, high performance liquid chromatography and liquid chromatography-mass spectrometry to verify the conversion product, and improves the screening efficiency of isoliquiritigenin conversion strains by gradually amplifying the detection accuracy. The method is not only efficient and reliable, but also provides a new technical idea and theoretical basis for the development of natural products and the application of microbial conversion. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Linear relationship of isoliquiritigenin solution

[0025] Figure 2 OD value difference between 0h and 48h of the mixture of the bacterial solution and the isoliquiritigenin solution.

[0026] Figure 3 TLC result of the mixture of the bacterial solution and the isoliquiritigenin solution cultured for 48h.

[0027] Figure 4 TLC result of the mixture of the bacterial solution and the isoliquiritigenin solution cultured for 0h.

[0028] Figure 5 HPLC result of the mixture of the bacterial solution and the isoliquiritigenin solution cultured for 48h.

[0029] Figure 6 HPLC result of the mixture of the bacterial solution and the isoliquiritigenin solution cultured for 0h.

[0030] Figure 7 Structural formula of daidzin.

[0031] Figure 8 Structural formula of luteolin.

[0032] Figure 9 Structural formula of benzyl 2-hydroxybenzoate.

[0033] Figure 10 UPLC-MS result of the mixture of the bacterial strain TRM19827 and the isoliquiritigenin solution for 0h.

[0034] Figure 11 UPLC-MS result of the mixture of the bacterial strain TRM19827 and the isoliquiritigenin solution for 48h.

[0035] Figure 12 UPLC-MS result of the mixture of the bacterial strain TRM19829 and the isoliquiritigenin solution for 0h.

[0036] Figure 13 UPLC-MS result of the mixture of the bacterial strain TRM19829 and the isoliquiritigenin solution for 48h. DETAILED DESCRIPTION

[0037] The present application will be described in detail below with reference to the drawings.

[0038] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and should not be used to limit the present application.

[0039] Example 1

[0040] In recent years, the use of microbial transformation technology to modify the structure of natural products has become an important way to develop new drugs and functional ingredients. Microbial transformation can efficiently and selectively modify the chemical structure of isofraxidin through specific enzymatic reactions, thereby enhancing its biological activity or improving its physicochemical properties. However, screening strains with high conversion capacity is a key step in this technology.

[0041] Traditional screening methods usually rely on a single analytical method, making it difficult to comprehensively evaluate the conversion efficiency and product diversity of the strains. Therefore, this embodiment adopts a multi-level analysis strategy combining a microplate reader, TLC, HPLC, and UPLC-MS to systematically screen isofraxidin conversion bacteria. The microplate reader can quickly screen the conversion of strains, TLC is used for re-screening flavonoid conversion bacteria to preliminarily identify the conversion products, HPLC provides high-resolution quantitative analysis, and UPLC-MS is used to accurately identify the molecular structure of the products. This multi-level screening method not only improves the screening efficiency, but also comprehensively analyzes the metabolic pathways during the conversion process, laying a foundation for subsequent functional optimization and application of the strains. This embodiment aims to quickly screen isofraxidin conversion strains through this method, providing new ideas and methods for the microbial transformation of natural products.

[0042] Experimental methods

[0043] Activation of strains

[0044] The bacterial strain stored in glycerol was inoculated into TSB medium at a 2% (v / v) inoculation amount and cultured in a constant temperature incubator at 37°C for 1-3 days. The fungal strain stored in glycerol was inoculated into CMM medium at a 2% (v / v) inoculation amount and cultured in a constant temperature incubator at 28°C for 5-7 days.

[0045] Preparation of control stock solution

[0046] Accurately weigh 5 g of isofraxidin powder and dissolve it in 5 mL of methanol solution. Store it in a 4°C refrigerator for standby use.

[0047] Screening of isofraxidin conversion bacteria by microplate reader

[0048] First, take 10 mL of activated bacterial solution in a 20 mL PA bottle, then add 500 μL of isofraxidin stock solution, and invert the mixture 3-5 times. Take 200 μL in a microplate and place it in the microplate reader for detection. The remaining mixture is placed in a shaking incubator at 180 r / min (bacteria at 37°C, fungi at 28°C) for 48 h. After incubation, repeat the above operation.

[0049] TLC screening of isofraxidin conversion bacteria

[0050] Dilute the liquor of isoliquiritigenin to 50 μg / mL, and prepare 20 mL of developing agent with the ratio of methanol: ethyl acetate: petroleum ether = 1:10:16. Use it to detect the control of isoliquiritigenin and the conversion product after the mixed culture of the bacteria liquor and isoliquiritigenin solution. Take 2-3 μL of the mixed liquor in step 4.2.3 and 2-3 μL of the control solution on the same silica gel GF 254 thin layer plate. After developing for 20 min, take it out and dry it with a hair dryer. Observe it under the dark box ultraviolet analyzer at 254 nm. Repeat the above operation after the mixed liquor culture.

[0051] HPLC verification of isoliquiritigenin conversion bacteria product

[0052] Select the strains with good effect screened by the microplate reader and TLC. Extract the mixed liquor of the bacteria liquor and isoliquiritigenin solution with ethyl acetate twice. Take the upper layer and use a syringe to take the upper liquid through a 0.22 μm microporous filter into a liquid phase vial for HPLC detection. The time elution program is shown in Table 1.

[0053] Table 1

[0054]

[0055] UPLC-MS verification of conversion product

[0056] Mix the bacteria liquor of strains TRM19827 and TRM19829 with isoliquiritigenin solution and culture for 48 h. After extraction and dilution, use the ultra-high performance liquid chromatography-mass spectrometer to detect it. The mobile phase A is water, the mobile phase B is methanol, and the injection amount is 10 μL. The specific information of the ultra-high performance liquid chromatography-mass spectrometer is as follows: the mobile phase used is methanol-water, the chromatographic column is C18 reverse phase chromatographic column (Waters ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 100 mm), the ion source temperature is 120℃ (ESI positive ion mode), the time program is 30 min, and the resolution is ≥100000FWHM (m / z 863).

[0057] Experimental results

[0058] Microplate reader screening results

[0059] In order to verify the reliability of the method, about 170 strains isolated in the laboratory were verified.

[0060] Determine the linear range of isoliquiritigenin as 5-100 μg / mL Figure 1 ) before the experiment. Then culture the mixed liquor of the bacteria liquor and isoliquiritigenin solution for 0 h, take 200 μL in the microplate, and place it in the microplate reader for detection. Repeat the above operation after the mixed liquor is cultured for 48 h Figure 2). The proposed MTHL method was also tested using about 1200 strains, and the results were satisfactory. It was found that the OD value after 48 h was lower than that at 0 h, and the OD difference of a few strains was higher than 0 h, and the OD difference was close when mixed with isoliquiritigenin and other flavonoids, which was consistent with our experimental results, which greatly helped to quickly screen isoliquiritigenin conversion bacteria. The experiment screened out 15 reliable strains, respectively TRM19771 (Bacillus massiliosenegalensis), TRM19773 (Penicillium polonicum), TRM19778 (Bacillus sp.), TRM19779 (Penicillium sp.), TRM19780 (Penicillium sp.), TRM19782 (Penicillium mononematosu), TRM19795 (Aspergillus ochraceus), TRM19796 (Penicillium roqueforti), TRM19797 (Penicillium paneum), TRM19798 (Penicillium roqueforti), TRM19800 (Fusarium solani), TRM19801 (Doratomyces sp.), TRM19802 (Fusarium solani), TRM19827 (Penicillium sp.), and TRM19829 (Penicillium crustosum). The experiment verified the strong conversion ability of fungi, and most bacteria did not have the ability to convert isoliquiritigenin.

[0061] TLC screening of isoliquiritigenin conversion bacteria

[0062] 2-3 μL of the mixture of bacterial solution and isoliquiritigenin solution and 2-3 μL of isoliquiritigenin control solution were transferred to the same silica gel plate into a centrifuge tube, and placed in the development tank for 20 min, and observed at 254 nm in the dark box of the ultraviolet spectrophotometer; the above operation was repeated 48 h after the mixed culture of bacterial solution and isoliquiritigenin solution. As shown in Figure 3 , the position of the mixture of bacterial solution and isoliquiritigenin solution on the thin layer plate after 48 h of culture was different from that of the standard solution, and no other substances were observed on the thin layer plate after mixing the culture medium solution with the isoliquiritigenin solution, indicating that the mixture produced other substances, and the culture medium did not interfere with the experiment, and the mixture of bacterial solution and isoliquiritigenin solution was also cultured for 0 h on the thin layer plate, and it could be seen from Figure 4 that no other substances were produced, providing a basis for subsequent experiments.

[0063] HPLC verification of the results of the transformation of isoliquiritigenin by the bacteria

[0064] After 0h of incubation of the mixture of bacteria and isoliquiritigenin solution, the mixture was extracted twice with ethyl acetate, the upper layer was removed, and the upper liquid was taken with a syringe and passed through a 0.22 μιη microporous filter into a liquid chromatography vial, which was then detected using high performance liquid chromatography. The above operation was repeated 48h after the incubation of the mixture of bacteria and isoliquiritigenin solution. Figure 5 It can be seen from Table 1 that the control bacteria and culture medium do not affect the peak of the standard substance, indicating that they do not affect the experimental results. Figure 6 It can be seen from Table 1 that the control bacteria and culture medium do not affect the peak of the standard substance, indicating that they do not affect the experimental results.

[0065] UPLC-MS verification of the results of the transformation of isoliquiritigenin by the bacteria

[0066] The bacteria of strains TRM19827 and TRM19829 were mixed with isoliquiritigenin solution and incubated for 48h, 1 mL of the mixture was taken, and after extraction and dilution, it was detected using UPLC-MS in the analysis and testing center. According to the chromatographic response value of the liquid chromatography-mass spectrometry instrument, some flavonoids were found (see Table 2). The results show that no isoliquiritigenin substance was found, which may be because the isoliquiritigenin was broken into other ion fragments during the UPLC-MS process, but other active flavonoid analogues were found, and TRM19829 may transform the product into two flavonoids, daidzein and luteolin, the structural formula is shown in Figure 7 and Figure 8 ; TRM19827 may transform the product into 2-hydroxybenzoic acid benzyl ester, the structural formula is shown in Figure 9 . Figure 10 and Figure 11 The detailed peak chart detected by UPLC-MS for TRM19827 is shown in Figure 12 and Figure 13 The detailed peak chart detected by UPLC-MS for TRM19829 is shown in

[0067] Table 2

[0068]

[0069] The components in Czapek's medium are NaNO3, K2HPO4, MgSO4·7H2O, KCl, FeSO4·7H2O and sucrose, and none of these components has the mother nucleus structure of flavonoids. Penicillium is a large class of fungi, which contains more than 1,300 metabolites, including azaphilones, cyclic peptides, steroids, alkaloids, terpenes and polyketides. These are the main compound types, and no literature directly indicates that the secondary metabolites of Penicillium are flavonoids.

[0070] In this embodiment, a multi-level analysis strategy combining microplate reader, TLC, HPLC and UPLC-MS is adopted, more than 170 strains are screened, and finally 15 high-efficiency isoflavone conversion strains are identified, including 9 Penicillium strains, 2 Bacillus strains, 2 Fusarium strains, 1 Aspergillus strain and 1 Doratomyces strain. The microplate reader screening conversion bacteria results show that the OD value of the mixed solution of the bacterial solution and the isoflavone solution is significantly reduced after 48 h, indicating that the isoflavone is effectively converted; TLC and HPLC further confirm the diversity of the conversion products, UPLC-MS detects that the possible conversion product of TRM19829 is benzyl 2-hydroxybenzoate, and the possible conversion product of TRM19827 is 2 flavonoids daidzein and luteolin. The experiment shows that fungi, especially Penicillium, play a dominant role in isoflavone conversion, while the conversion ability of bacteria is limited. The method provides an efficient screening method and strain resource for microbial conversion of natural products, and has important application potential.

[0071] The above only describes the preferred embodiments of the present application and should not be used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for rapid large-scale screening of isoliquiritigenin-transformed strains based on MTHM, characterized in that: The following steps are involved: Step 1: Mix the bacterial solution to be screened and the isoliquiritigenin solution to obtain a first mixed solution; use a microplate reader to detect the absorbance value of the first mixed solution; culture the first mixed solution for more than 48 hours to obtain a second mixed solution, and use a microplate reader to detect the absorbance value of the second mixed solution; the strain with an absolute value of the difference between the two absorbances greater than 0.4 is selected as the preliminary screened strain; Step 2: Using thin layer chromatography, the mixed solution of the bacterial solution of the initially screened strain and the isoliquiritigenin solution and the isoliquiritigenin standard product are respectively tested; the strains with different expansion positions in the mixed solution and the isoliquiritigenin standard product and the mixed solution having two or more expansion positions are selected as the strains screened for the second time; Step 3: Perform HPLC detection on the mixture of the bacterial solution of the strain screened for the second time and the isoliquiritigenin solution. If the peak of the mixed solution on the chromatogram shows other peaks next to the peak position of the isoliquiritigenin standard product or the peak type of the mixed solution is different from that of the standard product, the strain is considered to be the strain screened for the third time; Step 4: The bacterial liquid of the strain screened for the third time was mixed with the isoliquiritigenin solution and cultured for more than 48 hours, and the transformation products of the strain were detected under ultra-high performance liquid chromatography-mass spectrometry to screen out the final isoliquiritigenin-transformed strain.

2. The method for rapid large-scale screening of isoliquiritigenin-transformed strains based on MTHM according to claim 1, characterized in that: In step 1, the bacterial solution to be screened is obtained by strain activation treatment: When the strain is a bacterium, inoculate the strain into TSB medium and culture it in a constant temperature incubator at 36°C-38°C for 1-3 days; When the strain is a fungus, the strain is inoculated into CMM medium and cultured in a constant temperature incubator at 26° C.-30° C. for 5-7 days.

3. The method for rapid large-scale screening of isoliquiritigenin-transformed strains based on MTHM according to claim 1, characterized in that: In step 1, the first mixed solution is subjected to strain culture for 48 hours to 60 hours.

4. The method for rapid large-scale screening of isoliquiritigenin-transformed strains based on MTHM according to claim 1, characterized in that: In the step 1, the optimum concentration of the isoliquiritigenin standard solution detected by the microplate reader is 10-100 μg / mL.

5. The method for rapid large-scale screening of isoliquiritigenin-transformed strains based on MTHM according to claim 1, characterized in that: In the step 2, during the thin layer chromatography operation, the developing solvent is obtained by mixing the following raw materials in a volume ratio: methanol: ethyl acetate: petroleum ether of 1:8-12:15-16.

6. The method for rapid large-scale screening of isoliquiritigenin-transformed strains based on MTHM according to claim 1, characterized in that: In step 3, the mobile phase for HPLC detection is acetonitrile:methanol (9:1)-1% formic acid water.

7. The method for rapid large-scale screening of isoliquiritigenin-transformed strains based on MTHM according to any one of claims 1 to 6, characterized in that: In the step 4, the detection was performed using an ultra-high performance liquid chromatography-mass spectrometer, with mobile phase A being water, mobile phase B being methanol, and the injection volume being 10 μL.

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