Use of hungatella strain in biotransformation of flavone carbon glycoside

Abstract

The application discloses application of Hungatella strain in biological transformation of flavone carbon glycoside. The application provides a method for preparing aglycone or aglycone derivative, and comprises the following steps: taking glycoside or plant material containing glycoside as raw material, and adopting Hungatella strain or culture of Hungatella strain to perform biological transformation, so as to obtain aglycone or aglycone derivative. The method provided by the application is a microbial transformation method, and has the advantages of fast reaction rate, high product conversion rate, mild reaction condition, environmental friendliness, simple transformation process, short cycle, low cost and the like. The application is expected to efficiently hydrolyze carbon glycoside bond. The application has great application and promotion value in the field of production of aglycone or aglycone derivative and downstream products thereof.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and relates to application of Hungatella strain in biological transformation of flavone carbon glycoside. BACKGROUND

[0002] Flavonoids are a series of compounds in which two benzene rings are connected to each other through a central three-carbon chain. Flavone carbon glycoside is a C-glycoside in flavone glycoside. Flavone carbon glycoside is an important class of flavone glycoside, taking luteolin and apigenin as the parent body, the sugar group is directly connected to C-6 or C-8 through a C-C bond, such as vitexin, orientin, isovitexin, isoorientin, etc. Carbon glycoside compounds are stable in structure, and it is difficult to directly cleave the carbon glycoside bond by chemical method, and it is difficult to obtain the aglycone (such as luteolin and apigenin) or aglycone derivatives (such as dihydroluteolin, dihydroapigenin, etc.).

[0003] Luteolin and apigenin are natural flavonoids, which are widely distributed in plants. Luteolin has various pharmacological activities, such as anti-inflammatory, anti-allergic, uric acid-lowering, anti-tumor, antibacterial, anti-viral, etc.; apigenin has anti-inflammatory, anti-tumor, anti-spasm, anti-radiation effects, and can maintain bone health and protect the liver. Dihydroluteolin and dihydroapigenin are derivatives in which the C-2,3 double bond of luteolin and apigenin is reduced, respectively. Dihydroluteolin, also known as eriodictyol, has various pharmacological activities such as anti-oxidation, anti-inflammation, anti-tumor, neuroprotection, etc., and is often used for treating asthma, allergic rhinitis, rheumatism and other diseases; dihydroapigenin, also known as naringenin, has effects such as antibacterial, anti-inflammatory, free radical scavenging, anti-oxidation, cough relief and sputum elimination, blood lipid reduction, anti-cancer and anti-tumor, spasmolysis and cholagogue, prevention and treatment of liver disease, inhibition of platelet coagulation, and anti-atherosclerosis. SUMMARY

[0004] The present application aims to provide application of Hungatella strain in biological transformation of flavone carbon glycoside.

[0005] The present application provides application of Hungatella strain or culture of Hungatella strain in hydrolysis of carbon glycoside bond.

[0006] The present application also provides application of Hungatella strain or culture of Hungatella strain in hydrolysis of carbon glycoside bond of glycoside.

[0007] The present application also provides application of Hungatella strain or culture of Hungatella strain in preparation of aglycone or aglycone derivative.

[0008] The application also provides use of a Hungatella strain or a culture of a Hungatella strain in preparation of an aglycone or an aglycone derivative from a glycoside or a plant material containing a glycoside.

[0009] The application also provides use of a Hungatella strain or a culture of a Hungatella strain in conversion of a glycoside into an aglycone or an aglycone derivative.

[0010] The application also provides a method for preparing an aglycone or an aglycone derivative, comprising the following steps: using a Hungatella strain or a culture of a Hungatella strain to biotransform a glycoside or a plant material containing a glycoside, so as to obtain the aglycone or the aglycone derivative.

[0011] The application also provides a method for preparing luteolin and / or dihydro-luteolin, comprising the following steps: using a Hungatella strain or a culture of a Hungatella strain to biotransform isohydhn or a plant material containing isohydhn, so as to obtain the luteolin and / or dihydro-luteolin.

[0012] The application also provides use of a Hungatella strain or a culture of a Hungatella strain in preparation of luteolin and / or dihydro-luteolin.

[0013] The application also provides a method for preparing apigenin and / or dihydro-apigenin, comprising the following steps: using a Hungatella strain or a culture of a Hungatella strain to biotransform isoviolanthin or a plant material containing isoviolanthin, so as to obtain the apigenin and / or dihydro-apigenin.

[0014] The application also provides use of a Hungatella strain or a culture of a Hungatella strain in preparation of apigenin and / or dihydro-apigenin.

[0015] Illustratively, the culture of any of the Hungatella strains described above is a substance obtained by culturing the Hungatella strain.

[0016] Illustratively, the method for preparing the culture of any of the Hungatella strains described above is as follows: inoculating the Hungatella strain into a brain heart infusion (BHI) liquid medium, and culturing statically.

[0017] For example, the Hungatella strain can be Hungatella sp. DSM 11869.

[0018] For example, the Hungatella strain can be Hungatella sp. DSM 11869.

[0019] For example, the Hungatella strain can be Hungatella sp. DSM 11869.

[0020] For example, the carbonoside bond can be a carbonoside bond of a flavonoid carbonoside.

[0021] For example, the glycoside can be a glycoside of a glycoside compound.

[0022] For example, the glycoside can be a glycoside of a flavonoid carbonoside.

[0023] For example, the glycoside can be a glycoside of a flavonoid carbonoside.

[0024] For example, the aglycone can be a flavonoid carbonoside aglycone.

[0025] For example, the aglycone derivative can be a flavonoid carbonoside aglycone derivative.

[0026] For example, the glycoside can be isohymenose or isofukinol.

[0027] For example, the aglycone can be luteolin or apigenin.

[0028] For example, the aglycone derivative can be dihydroluteolin or dihydroapigenin.

[0029] For example, the biotransformation can be performed for 9-36 hours.

[0030] For example, the biotransformation can be performed for 9-36 hours.

[0031] For example, the biotransformation can be performed for 9-36 hours.

[0032] For example, the biotransformation can be performed for 9-36 hours.

[0033] For example, the biotransformation can be performed for 9-36 hours.

[0034] The term "culture" refers to the collective term for the liquid or solid product (all material in the culture vessel, i.e. the fermentation product) that has been inoculated and grown by man. That is, the product obtained by growing and / or amplifying microorganisms, which can be a biologically pure culture of microorganisms, or can contain certain amounts of culture medium, metabolites and / or other components produced during the cultivation. The term "culture" also includes the subcultures obtained by passing the microorganisms, which can be a culture of a certain generation, or a mixture of several generations.

[0035] The culture can be a microbial inoculum.

[0036] The method provided by the present application is microbial conversion, which has the advantages of fast reaction rate, high substrate conversion rate, mild reaction conditions, environmental friendliness, simple conversion process, short cycle and low cost. The present application is expected to efficiently hydrolyze carbon glycoside bonds. The present application has great application and promotion value in the field of production of aglycone or aglycone derivatives and downstream products thereof. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 LC / MS analysis results of luteolin in the conversion product of isohomosaligenin in Example 1.

[0038] Figure 2 LC / MS analysis results of dihydroluteolin in the conversion product of isohomosaligenin in Example 1.

[0039] Figure 3 Chromatogram of HPLC analysis of dihydroluteolin in the conversion product of isohomosaligenin in Example 1.

[0040] Figure 4 LC / MS analysis results of apigenin in the conversion product of isorhamnetin in Example 2.

[0041] Figure 5 LC / MS analysis results of dihydroapigenin in the conversion product of isorhamnetin in Example 2.

[0042] Figure 6 Chromatogram of HPLC analysis of dihydroapigenin in the conversion product of isorhamnetin in Example 2.

[0043] Figure 7 Chromatogram of separation and purification process in the preparation of dihydroluteolin in Example 3.

[0044] Figure 8 Chromatogram of HPLC analysis of the preparation product of dihydroluteolin in Example 3.

[0045] Figure 9 HPLC analysis of the preparation product of dihydroluteolin in Example 3. 1H-NMR analysis and 13 C-NMR analysis results.

[0046] Figure 10 Chromatogram for separation and purification process for preparing dihydroapigenin in Example 3.

[0047] Figure 11 Chromatogram for HPLC analysis of product for preparing dihydroapigenin in Example 3.

[0048] Figure 12 Chromatogram for HPLC analysis of product for preparing dihydroapigenin in Example 3. 1 H-NMR analysis and 13 C-NMR analysis results. DETAILED DESCRIPTION

[0049] The application will be further described in conjunction with the specific embodiments. The examples provided below are only for the purpose of illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0050] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0051] The quantitative tests in the following examples are all set up in triplicate, and the results are averaged, unless otherwise specified.

[0052] Hungatella sp. DSM 11869: strain numbered 11869 by Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; website: https: / / www.dsmz.de / ).

[0053] Isorhamnetin, molecular formula C 21 H 20 O 11 , CAS No. 4261-42-1, and the structural formula is shown in formula (I).

[0054]

[0055] Dihydroquercetin, molecular formula C 15 H 12 O6, CAS No. 552-58-9, and the structural formula is shown in formula (II).

[0056]

[0057] Quercetin, molecular formula C15 H 10 O6, CAS No. 491-70-3, having the structural formula (III).

[0058]

[0059] Isorhamnetin, molecular formula C 21 H 20 O 10 , CAS No. 29702-25-8, having the structural formula (IV).

[0060]

[0061] Dihydroapigenin, molecular formula C 15 H 12 O5, CAS No. 480-41-1, having the structural formula (V).

[0062]

[0063] Apigenin, molecular formula C 15 H 10 O5, CAS No. 520-36-5, having the structural formula (VI).

[0064]

[0065] BHI agar medium (pH 7.4 ± 0.2): Dried calf brain infusion powder 12.5 g / L, dried bovine heart infusion powder 5.0 g / L, D-glucose 2.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, disodium hydrogen phosphate 2.5 g / L, agar 15.0 g / L; the rest is water.

[0066] BHI liquid medium (pH 7.4 ± 0.2): Dried calf brain infusion powder 12.5 g / L, dried bovine heart infusion powder 5.0 g / L, D-glucose 2.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L, disodium hydrogen phosphate 2.5 g / L; the rest is water.

[0067] Unless otherwise specified, the culture conditions for static culture in the examples are as follows: temperature: 37°C; gas condition: N2 90%, H2 5%, CO2 5%.

[0068] Example 1, Hungatella sp. DSM 11869 biotransformation of isohomosaligenin

[0069] I. Establishment of microbial transformation process

[0070] 1. Hungatella sp. DSM 11869 was streaked on BHI agar medium and incubated for 3 days.

[0071] 2. After step 1, a single colony was picked and inoculated into a screw-capped test tube containing 10 mL of BHI liquid medium, and incubated for 16 hours to obtain a seed solution.

[0072] 3. 0.5 mL of the seed solution obtained in step 2 was inoculated into a screw-capped test tube containing 10 mL of BHI liquid medium containing isorhamnetin, and incubated to obtain a transformation product (the transformation product refers to the entire system after incubation). During incubation, samples were taken every 3 hours, and the total incubation time was 36 hours. The sample taken at time 0 was used as a control. The BHI liquid medium containing isorhamnetin was obtained by adding isorhamnetin to the BHI liquid medium; the concentration of isorhamnetin in the medium was set at 0.43 mM or 4.30 mM, respectively.

[0073] II. Qualitative analysis of the composition of the transformation product

[0074] 1 mL of the transformation product obtained in step 1 was added to 1 mL of water-saturated n-butanol, vortexed for 10 min, then centrifuged at 4000 rpm for 15 min, and the organic phase was collected. The remaining material was dissolved in 250 μL of methanol, and then subjected to LC / MS analysis.

[0075] The main chromatographic conditions are as follows:

[0076] Chromatographic column: Agilent XDB-C18 (4.6 x 150 mm, 5 μm);

[0077] Injection mode: direct injection by automatic injector; detection wavelength: 258 nm; column temperature: 35°C;

[0078] Mobile phase: A phase or B phase or a mixture of A phase and B phase; B phase: 0.2% (by volume) acetic acid in water; A phase: acetonitrile; flow rate of mobile phase: 1 mL / min;

[0079] Elution process: 0-5 min, A phase accounts for 10% by volume of the mobile phase, and the corresponding B phase accounts for 90% by volume of the mobile phase; 5-20 min, A phase increases linearly from 10% to 30% by volume of the mobile phase, and the corresponding B phase decreases linearly from 90% to 70% by volume of the mobile phase; 20-30 min, A phase increases linearly from 30% to 100% by volume of the mobile phase, and the corresponding B phase decreases linearly from 70% to 0% by volume of the mobile phase.

[0080] The main mass spectrometry conditions are as follows:

[0081] Ion source: ESI; scan range: 100-1000 m / z; ion source spray voltage: 4.5 kV; capillary voltage: 45 V; capillary temperature: 200 °C; sheath gas (nitrogen) flow rate: 40 units, sample introduction: direct injection from autosampler; detection mode: full scan positive and negative ion mass spectrometry.

[0082] LC / MS analysis was used to detect the formation of luteolin and dihydroluteolin in the biotransformation products of isohamnetin by Hungatella sp. DSM 11869. The identification of luteolin in the biotransformation products of isohamnetin by Hungatella sp. DSM 11869 (treatment group with isohamnetin concentration of 0.43 mM, incubation time of 3 hours) is shown in Figure 2A (LC chromatogram, arrow shows target product peak) and Figure 2B (mass spectrum in positive ion mode, arrow shows quasi-molecular ion peak). Figure 1 (A: mass spectrum in positive ion mode, arrow shows quasi-molecular ion flow; B: mass spectrum in negative ion mode, arrow shows quasi-molecular ion peak). The identification of dihydroluteolin in the biotransformation products of isohamnetin by Hungatella sp. DSM 11869 (treatment group with isohamnetin concentration of 0.43 mM, incubation time of 12 hours) is shown in Figure 3A (LC chromatogram, arrow shows target product peak) and Figure 3B (mass spectrum in positive ion mode, arrow shows quasi-molecular ion peak). Figure 2 (A: LC chromatogram, arrow shows target product peak; B: mass spectrum in positive ion mode, arrow shows quasi-molecular ion peak). As shown in the figure: ESI m / z 287.27 [M+H] + and m / z 285.19 [M-H] － is the quasi-molecular ion peak of luteolin, and the product is the carbon glycoside aglycone obtained after C-deglycosylation of isohamnetin; ESI m / z 289.23 [M+H] + is the quasi-molecular ion peak of dihydroluteolin, and the product is the derivative obtained after C-deglycosylation and reduction of the double bond at C-2,3 of isohamnetin.

[0083] III. Quantitative analysis of microbial transformation efficiency

[0084] Take 1 mL of the transformation product obtained in step one, add 1 mL of water-saturated n-butanol, vortex for 10 min, then centrifuge at 4000 rpm for 15 min, collect the organic phase, concentrate under reduced pressure at 38 °C to remove the solvent, dissolve the residue in 250 μL of methanol, then perform HPLC analysis.

[0085] The HPLC parameters are as follows:

[0086] Chromatographic column: Agilent XDB-C18 (4.6 x 150 mm, 5 μm);

[0087] Detection wavelength: 258 nm; column temperature: 35 °C.

[0088] Mobile phase: A phase or B phase or mixture of A phase and B phase; B phase is 0.2% (volume ratio) acetic acid aqueous solution; A phase is acetonitrile; flow rate of mobile phase: 1 mL / min;

[0089] Elution process: 0-5 min, volume fraction of A phase in mobile phase is 10%, and volume fraction of corresponding B phase in mobile phase is 90%; 5-20 min, volume fraction of A phase in mobile phase is linearly increased from 10% to 30%, and volume fraction of corresponding B phase in mobile phase is linearly decreased from 90% to 70%; 20-30 min, volume fraction of A phase in mobile phase is linearly increased from 30% to 100%, and volume fraction of corresponding B phase in mobile phase is linearly decreased from 70% to 0%.

[0090] The relative conversion rate of the substrate is calculated by peak area normalization method;

[0091] Relative conversion rate = product peak area / (substrate peak area + product peak area) * 100%;

[0092] The product peak is the peak corresponding to dihydroxylin; the substrate peak is the peak corresponding to isohomosaligenin.

[0093] The yield of dihydroxylin in the conversion product is calculated by external standard point method.

[0094] When the concentration of isohomosaligenin is 0.43 mM: after 6 hours of biotransformation, 1 / 5 of the substrate is converted; after 9 hours of biotransformation, the conversion rate of isohomosaligenin is 50%; after 12 hours of biotransformation, the conversion rate of isohomosaligenin reaches 99%, and the yield of dihydroxylin in the conversion product is not less than 110 mg / L, basically all isohomosaligenin in the culture medium is converted into the corresponding product. When the concentration of isohomosaligenin is 4.30 mM: after 36 hours of biotransformation, the yield of dihydroxylin in the conversion product is close to 280 mg / L. The results show that Hungatella sp. DSM 11869 has good biotransformation activity on isohomosaligenin.

[0095] The HPLC analysis chromatogram of biotransformation (treatment group with the concentration of isohomosaligenin in the culture medium set to 0.43 mM) is shown in Figure 3 . Figure 3 A is the chromatogram of isohomosaligenin standard; B is the chromatogram of the substrate at 0 time, and the arrow indicates isohomosaligenin; C is the chromatogram of the conversion product after 24 hours of conversion, and the arrow indicates dihydroxylin.

[0096] Example 2, biotransformation of isohomosaligenin by Hungatella sp. DSM 11869

[0097] I. Establishment of microbial conversion process

[0098] Replacing isoharman with isohamnetoxin, otherwise same as step one of example 1.

[0099] II. Qualitative analysis of the composition of the transformation products

[0100] Method same as step two of example 1.

[0101] LC / MS analysis was used to detect the formation of apigenin and dihydroapigenin in the biotransformation products of isohamnetoxin by Hungatella sp. DSM 11869. The identification of apigenin in the biotransformation products of isohamnetoxin by Hungatella sp. DSM 11869 (treatment group with isohamnetoxin concentration set at 0.43 mM, incubation time of 3 hours) is shown in Figure 1. Figure 4 (A: mass spectrum in positive ion mode, arrow shows the molecular ion flow; B: mass spectrum in negative ion mode, arrow shows the molecular ion peak). The identification of dihydroapigenin in the biotransformation products of isohamnetoxin by Hungatella sp. DSM 11869 (treatment group with isohamnetoxin concentration set at 0.43 mM, incubation time of 12 hours) is shown in Figure 2. Figure 5 (A: LC chromatogram, arrow shows the target product peak; B: mass spectrum in positive ion mode, arrow shows the molecular ion peak). As shown in the figure: ESI m / z 271.07 [M+H] + and m / z 269.07 [M-H] － is the molecular ion peak of apigenin, which is the carbon glycoside aglycone obtained after C-deglycosylation of isohamnetoxin; ESI m / z 273.10 [M+H]+is the molecular ion peak of dihydroapigenin, which is the derivative obtained after C-deglycosylation and reduction of the double bond at C-2,3 of isohamnetoxin.

[0102] III. Quantitative analysis of the efficiency of microbial transformation

[0103] Method same as step three of example 1.

[0104] The relative conversion rate of the substrate was calculated by peak area normalization method;

[0105] Relative conversion rate = product peak area / (substrate peak area + product peak area) * 100%;

[0106] The product peak corresponds to the peak of dihydroapigenin; the substrate peak corresponds to the peak of isohamnetoxin.

[0107] The yield of dihydroapigenin in the transformation products was calculated by external standard one-point method.

[0108] When the concentration of isorhamnetin is 0.43 mM: after 9 hours of biotransformation, the conversion rate of isorhamnetin is 80%; after 12 hours of biotransformation, the conversion rate of isorhamnetin reaches 99%, and the yield of dihydroxyarnetin in the conversion product is not less than 100 mg / L, and basically all the isorhamnetin in the culture medium is converted into the corresponding product. When the concentration of isorhamnetin is 4.30 mM: after 36 hours of biotransformation, the yield of dihydroxyarnetin in the conversion product is close to 260 mg / L. The intestinal bacteria Hungatella sp. DSM 11869 has good biotransformation activity on isorhamnetin.

[0109] The HPLC analysis chromatogram of biotransformation (the treatment group with the concentration of isorhamnetin in the culture medium set to 0.43 mM) is shown in Figure 6 . Figure 6 A is the chromatogram of isorhamnetin standard; B is the chromatogram of the substrate at 0 time, and the arrow indicates isorhamnetin; C is the chromatogram of the conversion product after 24 hours of conversion, and the arrow indicates dihydroxyarnetin.

[0110] Example 3, Preparation of flavonoid carbon glycoside biotransformation product by Hungatella sp. DSM 11869

[0111] I. Preparation of dihydroxyarnetin

[0112] 1. Biotransformation of isohomosaligenin by Hungatella sp. DSM 11869

[0113] (1) Hungatella sp. DSM 11869 was streaked on BHI agar medium and incubated for 3 days.

[0114] (2) After completing step (1), a single colony was picked and inoculated into a screw-capped test tube containing 10 mL of BHI liquid medium, and incubated for 16 hours to obtain a seed solution.

[0115] (3) 2.5 mL of the seed solution obtained in step (2) was inoculated into a screw-capped test tube containing 50 mL of BHI liquid medium containing isohomosaligenin, and incubated for 24 hours to obtain the conversion product (the conversion product refers to the entire system after incubation). The BHI liquid medium containing isohomosaligenin is obtained by adding isohomosaligenin to the BHI liquid medium; the concentration of isohomosaligenin in the culture medium is set to 0.43 mM.

[0116] Through multiple repeated treatments, 750 mL of conversion product was obtained.

[0117] 2. Separation and purification of biotransformation product

[0118] Take 750 mL of the conversion product prepared in Step 1, extract with ethyl acetate and collect the ethyl acetate phase (3 times extraction, each time add 3 times volume of ethyl acetate, collect the ethyl acetate phase after extraction; combine the three times collected ethyl acetate phase), concentrate under reduced pressure at 38°C to remove the solvent, dissolve the residue in 10 mL of 70% (volume percent) methanol aqueous solution, then filter with 0.45 μm microporous filter and collect the filtrate.

[0119] Take the filtrate, separate and purify the target product by preparative medium pressure liquid chromatography (MPLC).

[0120] The chromatographic parameters are as follows:

[0121] Chromatograph: Jiangsu Hanbang NS4000 series preparative medium pressure liquid chromatograph;

[0122] Chromatographic column: Green grass preparative SilGreen-C18 chromatographic column (250 x 4.6 mm, 5 μm);

[0123] Mobile phase: composed of 40 volume parts of methanol, 0.2 volume parts of acetic acid and 59.8 volume parts of water;

[0124] Flow rate of mobile phase: 4 mL / min; injection volume: 1 mL.

[0125] The chromatogram of the separation and purification process is shown in Figure 7 , and the arrow indicates the target peak.

[0126] Collect the solution after the column corresponding to the target peak, concentrate under reduced pressure at 38°C to remove the solvent, and obtain the powder product.

[0127] After 750 mL of the conversion product prepared in Step 1 is subjected to Step 2, about 68 mg of powder product is obtained.

[0128] 3. Chromatographic analysis of the product prepared

[0129] The methanol solution of the powder product obtained in Step 2 is subjected to HPLC analysis (parameters same as those in Step 3 of Example 1), and the results are shown in Figure 8 , and the arrow indicates the target product.

[0130] 4. Structural identification of the product prepared

[0131] The dimethyl sulfoxide-d6 (DMSO-d6) solution of the powder product obtained in Step 2 is subjected to 1 H-NMR analysis and 13 C-NMR analysis, and the results are shown in Figure 9 . Figure 9 Medium: A is 13 C-NMR spectrum, and B is 1H-NMR spectrum. The results are consistent with the NMR data of the known compound, dihydroquercetin, which has been reported.

[0132] Preparation of dihydroapigenin

[0133] 1. Bioconversion of apigenin by Hungatella sp. DSM 11869

[0134] Apigenin was replaced by isohomosaligenin, and other procedures were the same as 1 of Step 1.

[0135] 2. Separation and purification of the bioconversion product

[0136] The method was basically the same as 2 of Step 1, except that the composition of the mobile phase was different.

[0137] The mobile phase consisted of 50 parts by volume of methanol, 0.2 parts by volume of acetic acid and 49.8 parts by volume of water.

[0138] The chromatogram of the separation and purification process is shown in Figure 10 , and the arrow indicates the target peak.

[0139] The solution after passing through the column corresponding to the target peak was collected, and concentrated under reduced pressure at 38°C to remove the solvent to obtain a powdery product.

[0140] After 750 mL of the conversion product prepared in Step 1 was subjected to Step 2, about 64 mg of a powdery product was obtained.

[0141] 3. Chromatographic analysis of the prepared product

[0142] The methanol solution of the powdery product obtained in Step 2 was subjected to HPLC analysis (the parameters were the same as those in Step 3 of Example 1), and the results are shown in Figure 11 , and the arrow indicates the target product.

[0143] 4. Structural identification of the prepared product

[0144] The DMSO-d6 solution of the powdery product obtained in Step 2 was subjected to 1 H-NMR analysis and 13 C-NMR analysis, and the results are shown in Figure 12 . Figure 12 A is 13 the C-NMR spectrum, and B is 1 the H-NMR spectrum. The results are consistent with the NMR data of the known compound, dihydroquercetin, which has been reported.

[0145] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a specific example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Hungatella the use of a strain of the genus lachancea in the preparation of an aglycone or aglycone derivative from a glycoside or glycoside-containing plant material; The Hungatella Strain is Hungatella sp. DSM 11869; the glycoside is isohamnetin or isojaceosidin; the aglycone is luteolin or apigenin; the aglycone derivative is dihydroluteolin or dihydroapigenin.

2. Hungatella Use of a culture of a strain of the genus Agaricus in the preparation of an aglycone or aglycone derivative from a glycoside or glycoside-containing plant material; The Hungatella Strains belonging to the species Lactobacillus Hungatella sp. DSM 11869; The Hungatella The method for preparing a culture of the strain of the genus Brucella is to inoculate the strain of the genus Brucella into a brain heart infusion liquid medium and incubate it statically. Hungatella The method for preparing a culture of the strain of the genus Brucella is to inoculate the strain of the genus Brucella into a brain heart infusion liquid medium and incubate it statically. the glycoside is isohamnetin or isojaceosidin; the aglycone is luteolin or apigenin; the aglycone derivative is dihydroluteolin or dihydroapigenin.

3. Hungatella the use of a strain of the genus Lactobacillus in the conversion of a glycoside into an aglycone or aglycone derivative; The Hungatella Strains belonging to the species Lactobacillus Hungatella sp. DSM 11869; the glycoside is isohamnetin or isojaceosidin; the aglycone is luteolin or apigenin; the aglycone derivative is dihydroluteolin or dihydroapigenin.

4. Hungatella Use of a culture of a strain of the genus Agrobacterium in the conversion of a glycoside into an aglycone or aglycone derivative; The Hungatella Strains belonging to the species Lactobacillus Hungatella sp. DSM 11869; The Hungatella The method for preparing a culture of the strain of the genus Brucella is to inoculate the strain of the genus Brucella into a brain heart infusion liquid medium and incubate it statically. Hungatella The method for preparing a culture of the strain of the genus Brucella is to inoculate the strain of the genus Brucella into a brain heart infusion liquid medium and incubate it statically. the glycoside is isohamnetin or isojaceosidin; the aglycone is luteolin or apigenin; the aglycone derivative is dihydroluteolin or dihydroapigenin.

5. A method for preparing luteolin and / or dihydroluteolin, comprising the steps of: using isohamnetin or plant material containing isohamnetin as raw material, using strain of genus Aspergillus for biotransformation to obtain luteolin and / or dihydroluteolin. Hungatella Aspergillus. The Hungatella Strain is Hungatella sp. DSM 11869.

6. A method for preparing luteolin and / or dihydroluteolin, comprising the steps of: using isohamnetin or plant material containing isohamnetin as raw material, using the culture of a strain belonging to the genus Aspergillus for bioconversion to obtain luteolin and / or dihydroluteolin. Hungatella a strain belonging to the genus Aspergillus for bioconversion to obtain luteolin and / or dihydroluteolin. The Hungatella Strains belonging to the species Lactobacillus Hungatella sp. DSM 11869; The Hungatella The method for preparing a culture of a strain of the genus Legionella is to inoculate the strain of the genus Legionella into a brain heart infusion liquid medium and to incubate it statically. Hungatella The method for preparing a culture of a strain of the genus Legionella is to inoculate the strain of the genus Legionella into a brain heart infusion liquid medium and to incubate it statically.

7. A method for preparing apigenin and / or dihydroapigenin, comprising the steps of: using isovertril or plant material containing isovertril as raw material, using the strain of Aspergillus shirousami to carry out bioconversion, and obtaining apigenin and / or dihydroapigenin. Hungatella Aspergillus shirousami. The Hungatella Strain is Hungatella sp. DSM 11869.

8. A method for preparing apigenin and / or dihydroapigenin, comprising the steps of: using isovitexin or plant material containing isovitexin as raw material, using the culture of a strain belonging to the genus Aspergillus for bioconversion, and obtaining apigenin and / or dihydroapigenin. Hungatella Aspergillus The Hungatella Strain is Hungatella sp. DSM 11869; The Hungatella The method for preparing a culture of the strain of the genus Brucella is to inoculate the strain of the genus Brucella into a brain heart infusion liquid medium and incubate it statically. Hungatella The method for preparing a culture of the strain of the genus Brucella is to inoculate the strain of the genus Brucella into a brain heart infusion liquid medium and incubate it statically.

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