An oxygen-tolerant Blautiae strain and its application in aerobic synthesis of 3′-demethylarcigenin

Abstract

The present invention relates to the field of microbial technology, and specifically discloses an oxygen-resistant Blautia sp. and its application in the aerobic synthesis of 3'-demethyl arctiin. The oxygen-resistant Blautia sp. (Blautia sp.) Aeroto-AUH-df6 has a deposit number of CGMCC NO.30297. The oxygen-resistant Blautia sp. Aeroto-AUH-df6 provided by the present invention can efficiently transform arctiin under the condition of air oxygen, and the transformation performance is stable, which solves the problem that 3'-demethyl arctiin cannot be biosynthesized on a large scale under the condition of air oxygen, and the problem that 3'-demethyl arctiin resources are scarce, and 3'-demethyl arctiin is the only product of the transformation of the substrate arctiin by the strain Aeroto-AUH-df6, which greatly simplifies the subsequent separation and purification steps, and is of great significance for studying the pharmacological activity of 3'-demethyl arctiin and the development of new drugs.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to an oxygen-resistant Blautiae bacterium and application thereof in aerobic synthesis of 3'-demethyl arctigenin. Background Art

[0002] Burdock (Arctium lappa L.) is a biennial herbaceous plant of the genus Arctium in the Asteraceae family. Burdock seeds are the dried mature fruits of burdock and are traditional medicines in my country. Burdock seeds are bitter and cold in nature. They belong to the lung and stomach meridians and have the effects of clearing heat, reducing swelling, detoxifying, exuding rash, and relieving sore throat. In addition, the "Compendium of Materia Medica" first recorded that burdock seeds can "facilitate defecation and urination", and this function is reflected in the "Compendium of Materia Medica" and "Compendium of Materia Medica Congxin". The results of modern pharmacological studies have shown that burdock seeds have multiple effects such as anti-inflammatory, antiviral, antioxidant, anti-diabetic, antitussive, hypolipidemic, anti-cancer, immune activity, anti-nephropathy, and liver protection. Burdock seeds mainly contain lignans, volatile oils, phenolic hydroxyl and thioacetylene compounds, etc., and also contain a small amount of alkaloids, sterols and vitamins. Among them, the largest content is the lignan compound arctiin, which is the iconic component of burdock seeds. The content of arctiin in the Chinese medicine burdock is 5.3% to 7.3%. Under oral conditions, arctiin can be hydrolyzed by glycosidase produced by intestinal flora to form arctigenin, which then exerts its therapeutic effect. Existing research results show that arctigenin can be converted into various metabolites by human fecal flora.

[0003] In 1992, Japanese scholar Nose et al. found that the intestinal flora of mice can convert arctiin into two metabolites, one of which is arctigenin and the other is 3′-demethyl arctigenin (3′-DMAG). In 2007, Japanese scholar Jin et al. isolated for the first time from fresh human feces a strain of anaerobic true bacteria Eubacterium sp.ARC-2 that can simultaneously convert the substrate arctiin into seven different metabolites, one of which is 3′-demethyl arctigenin. Although Jin et al. isolated a single bacterial strain with the function of converting arctiin, the strain generated a variety of products, resulting in a limited amount of each product synthesized. In addition, the high-performance liquid phase retention time of the product peaks was close, making separation and purification difficult. In 2011, Li Furui et al. reported an exploratory study on the artificial synthesis of 3′-DMAG by chemical methods. Although 3′-DMAG was eventually obtained, pyridine with a foul odor and strong irritation needed to be added during the reaction process. The synthesis process was harmful to human health and caused pollution to the environment. Therefore, it is currently impossible to achieve large-scale production of 3′-DMAG by chemical synthesis methods. In recent years, researchers have found that trace amounts of 3′-DMAG exist naturally in the traditional Chinese medicine Trachelospermum schrenkiana, and extracted and purified it. Due to its extremely low content, the extracted 3′-DMAG is expensive (3,000 yuan / 5 mg). In 2013, the inventors' team isolated a strictly anaerobic Blautia sp. strain AUH-JLD56 from human feces. The strain can efficiently convert arctiin or arctigenin into 3′-DMAG under anaerobic conditions, and 3′-DMAG is the only product generated by the strain AUH-JLD56 after converting the substrate arctigenin. In the anaerobic workstation, the maximum concentration of arctigenin that strain AUH-JLD56 can efficiently convert is 3.6 mmol / L, and the average conversion rate is 90.5%. Since strain Blautia sp.AUH-JLD56 is a strict anaerobic bacterium, it can only be inoculated and complete the conversion of the substrate arctigenin under strict anaerobic conditions, and maintaining an anaerobic environment for a long time requires a large amount of capital investment and is costly. Summary of the invention

[0004] In view of the above problems existing in the existing 3′-demethyl arctiin conversion or synthesis process, the present invention provides an oxygen-resistant Blautiae Aeroto-AUH-df6 and its application in the aerobic synthesis of 3′-demethyl arctiin. The oxygen-resistant Blautiae Aeroto-AUH-df6 provided by the present invention can efficiently convert the substrate arctiin into 3′-demethyl arctiin under natural conditions (i.e., under air oxygen conditions), and the conversion performance is stable, which solves the problem that 3′-demethyl arctiin cannot be biosynthesized on a large scale under air oxygen conditions, and has a great promoting effect on the pharmacological activity research of 3′-demethyl arctiin and the development of new drugs.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] The invention discloses an oxygen-resistant Blautia sp. Aeroto-AUH-df6, whose deposit number is CGMCCNO.30297.

[0007] The present invention performs oxygen-resistance acclimation on strictly anaerobic bacteria Blautia sp.AUH-JLD56 (CGMCC NO.8032) to obtain an oxygen-resistant mutant strain with high-efficiency conversion ability of the substrate arctigenin under the condition of air oxygen, separates and identifies the strain, and classifies and names the strain as oxygen-resistant Blautia sp., and its 16S rRNA gene sequence is as shown in SEQ ID NO.3, which has been deposited in the General Microbiology Center of China National Committee for the Preservation of Microorganisms and Cultures, referred to as CGMCC, on April 10, 2024, with a strain collection number of CGMCC NO.30297, and a preservation address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The aerobic Blautia sp. Aeroto-AUH-df6 provided by the present invention can grow normally under aerobic conditions with a BHI liquid culture medium height of 4 cm and an inoculation amount of not less than 1.25%. 600 The average is 2.2. The maximum conversion concentration of arctiin in an ordinary biochemical incubator is 8.2mmol / L, the average conversion rate is 90.0%, and the conversion performance is stable, which effectively solves the problem that 3′-demethyl arctiin cannot be biosynthesized on a large scale under air oxygen conditions. It has a great promoting effect on the pharmacological activity research of 3′-demethyl arctiin and the development of new drugs.

[0009] The pathway for microbial conversion of arctigenin to 3′-demethylarcigenin is as follows:

[0010]

[0011] The taxonomic characteristics of the aerotolerant Blautiae Aeroto-AUH-df6 of the present invention are:

[0012] The colony diameter is 6-7mm, milky white, with irregular edges, slightly white in the center but no obvious protrusion, smooth and moist; in BHI liquid medium, the bacteria are short rods with septa in the middle. The strain is Gram-positive; the strain is urease-positive and can utilize most carbohydrates, such as sucrose, sucrose, xylose, arabinose, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose and trehalose; catalase-negative, indole reaction-negative.

[0013] The present invention also provides the use of the aerobic Blautiae Aeroto-AUH-df6 in aerobic transformation of arctiin to synthesize 3'-demethyl arctiin.

[0014] Previous research results by the inventors' team have shown that at the same concentration, the product 3′-demethyl arctigenin has a significantly higher ability to scavenge DPPH free radicals (p<0.01) than the substrate arctigenin. In addition, the inventors' team collaborated with China Agricultural University to study the anticancer activity of 3′-demethyl arctigenin and found that compared with arctigenin, 3′-demethyl arctigenin has a stronger inhibitory effect on human liver cancer cells Hep G2, and is effective against liver cancer in vitro and in mice (Zhang E et al., Phytotherapy Res 2019).

[0015] The oxygen-resistant Blautiae Aeroto-AUH-df6 provided by the present invention can efficiently and stably transform arctigenin under the condition of air oxygen, and 3′-demethyl arctigenin is the only product of the strain Aeroto-AUH-df6 transforming the substrate arctigenin, which greatly simplifies the subsequent separation and purification steps, and is of great significance for studying the pharmacological activity of 3′-demethyl arctigenin and developing new drugs.

[0016] The present invention also provides a method for synthesizing 3'-demethyl arctiin, comprising the following steps: using arctiin as a substrate, using the culture of the oxygen-tolerant Blautiae Aeroto-AUH-df6 to transform under the condition of air oxygen to obtain 3'-demethyl arctiin.

[0017] Furthermore, the method for synthesizing 3′-demethyl arctigenin specifically comprises the following steps:

[0018] The seed liquid of aerotolerant Blautiae Aeroto-AUH-df6 is inoculated into a culture medium, arctiin mother liquid is added, and the culture is carried out at a constant temperature under the condition of air oxygen. After the transformation is completed, extraction, separation and purification are performed to obtain 3'-demethyl arctiin.

[0019] Furthermore, the culture medium is fresh BHI liquid culture medium.

[0020] Preferably, the inoculation amount of the seed solution of the aerotolerant Blautiae Aeroto-AUH-df6 is 5% to 10% (v / v).

[0021] Preferably, the concentration of the arctigenin mother solution is 200 mmol / L to 250 mmol / L.

[0022] It should be noted that the arctigenin mother solution is obtained by first adding a co-solvent dimethyl sulfoxide (DMSO) to arctigenin until it is completely dissolved, and then adding 100% methanol solvent to the above-mentioned mother solution concentration.

[0023] Preferably, the final concentration of arctigenin in the culture medium is 5 mmol / L to 10 mmol / L.

[0024] It should be noted that in order to ensure the rapid proliferation of aerotolerant Blautiae Aeroto-AUH-df6, arctigenin mother solution was added to the culture medium several times.

[0025] As a specific embodiment of the present invention, the specific steps of adding arctigenin mother solution are:

[0026] After inoculating the seed solution of Aeroto-AUH-df6, 1.0 mL to 2.0 mL of arctiin mother solution was added to 200 mL of BHI liquid culture medium for the first time, so that the initial concentration of arctiin in the culture medium was 1.0 mmol / L to 2.0 mmol / L. After culturing in a constant temperature incubator for 5 h to 10 h, 1.0 mL to 2.0 mL of arctiin mother solution was added. The method was supplemented 4 times in total, so that the total concentration of arctiin in the culture medium was 5 mmol / L to 10 mmol / L. Finally, the culture was co-cultured in a constant temperature incubator for 2 d to 3 d.

[0027] Preferably, the constant temperature culture temperature is 36° C. to 38° C., and the constant temperature culture time is 2 days to 3 days.

[0028] Furthermore, the separation and purification specifically includes the following steps: adding ethyl acetate to the culture for extraction, filtering the extract and evaporating it to dryness, adding 100% methanol to dissolve it, filtering it and performing semi-preparative high performance liquid chromatography separation, collecting the eluate with the product peak, evaporating it to dryness, and obtaining a pure product of 3′-demethylarcitogenin.

[0029] As a specific implementation of the present invention, the evaporation temperature is 50°C to 60°C.

[0030] As a specific implementation of the present invention, the extraction is performed 2 to 3 times.

[0031] Preferably, the semi-preparative HPLC separation conditions are: Chromatographic column: Kromasil C 18 Preparation column, 250mm×10mm, 5μm; a mixed solution of acetonitrile and acetic acid aqueous solution in a volume ratio of 8-12:92-88, mobile phase B: a mixed solution of acetic acid aqueous solution and acetonitrile in a volume ratio of 8-12:92-88, wherein the volume concentration of the acetic acid aqueous solution is 0.08%-0.12%; isocratic elution, mobile phase A:mobile phase B=58-62:42-38 (v / v); flow rate: 1.8mL / min-2.2mL / min; injection volume: 80μL-150μL; detection wavelength: 278nm-282nm.

[0032] More preferably, the semi-preparative HPLC separation conditions are: Chromatographic column: Kromasil C 18 Preparative column, 250mm×10mm, 5μm; mobile phase A: 10% acetonitrile-90% acetic acid aqueous solution, mobile phase B: 10% acetic acid aqueous solution-90% acetonitrile, the volume concentration of acetic acid aqueous solution is 0.1%; isocratic elution, mobile phase A: mobile phase B = 60:40 (v / v); flow rate: 2.0mL / min; injection volume: 120μL; detection wavelength: 280nm.

[0033] Furthermore, the preparation method of the seed liquid of the oxygen-resistant Blautiae Aeroto-AUH-df6 comprises the following steps: thawing the frozen oxygen-resistant Blautiae Aeroto-AUH-df6, inoculating it into a BHI liquid culture medium at an inoculation rate of 15% to 20% (v / v), and culturing it at 36° C. to 38° C. for 20 h to 25 h to obtain the activation liquid of the oxygen-resistant Blautiae Aeroto-AUH-df6; inoculating the activation liquid into a BHI culture medium at an inoculation rate of 5% to 10% (v / v), and culturing it at 36° C. to 38° C. for 12 h to 18 h to obtain the seed liquid of the oxygen-resistant Blautiae Aeroto-AUH-df6.

[0034] As a specific embodiment of the present invention, the culture can be carried out in a common biochemical incubator.

[0035] The maximum concentration of arctiin, a substrate, that can be efficiently converted by untamed strict anaerobic Blautiae AUH-JLD56 (CGMCC NO.8032) in an anaerobic workstation is 3.6 mmol / L. The maximum concentration of arctiin, a substrate, that can be efficiently converted by the aerobic Blautiae Aeroto-AUH-df6 provided by the present invention in the presence of air oxygen is 8.2 mmol / L, and the maximum conversion capacity is nearly 2.3 times that of the untamed original strain AUH-JLD56 in the anaerobic workstation.

[0036] The oxygen-resistant Blautiae Aeroto-AUH-df6 provided by the present invention can efficiently convert arctiin under the condition of air oxygen, and the conversion performance is stable. The method for synthesizing 3′-demethyl arctiin by using the oxygen-resistant Blautiae Aeroto-AUH-df6 is simple, which solves the problems that 3′-demethyl arctiin cannot be biosynthesized on a large scale under the condition of air oxygen and the shortage of 3′-demethyl arctiin resources, is conducive to reducing the synthesis cost of 3′-demethyl arctiin, and has a great promoting effect on the pharmacological activity research of 3′-demethyl arctiin and the development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The phylogenetic tree of Aeroto-AUH-df6 based on 16S rDNA sequence is shown in Figure 2.

[0038] Figure 2 is a high performance liquid chromatogram of the transformation of the substrate arctigenin by the aerotolerant Blautiae Aeroto-AUH-df6 in Example 1 of the present invention;

[0039] Figure 3 The UV absorption spectrum of the substrate arctigenin and the generated product 3′-demethylarcigenin transformed by the aerotolerant Blautiae Aeroto-AUH-df6 in Example 1 of the present invention;

[0040] Figure 4 The mass spectrum of the product obtained by converting the substrate arctigenin by Aeroto-AUH-df6 in Example 1 of the present invention;

[0041] Figure 5 The figure is a conversion dynamic curve diagram of the substrate arctigenin converted by the aerotolerant Blautiae Aeroto-AUH-df6 in Example 1 of the present invention;

[0042] Figure 6 This is a comparison chart of the conversion capabilities of the aerotolerant Blautiae Aeroto-AUH-df6 in Example 1 of the present invention to arctigenin at different concentrations. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] The BHI medium (1000 mL) used in the following examples was composed of: 12.5 g brain infusion solids, 5.0 g beef heart infusion solids, 10.0 g protease peptone, 2.0 g glucose, 5.0 g sodium chloride, and 2.5 g disodium hydrogen phosphate.

[0045] The composition of the acclimation medium used in the following examples is: 0.08% (m / v) agar, 0.15% (m / v) L-cysteine ​​and 0.15% (m / v) ascorbic acid are added to BHI liquid medium.

[0046] The methanol used in the following examples is all commercially available chromatography grade methanol, and the arctigenin mother solution is prepared by dissolving arctigenin in DMSO and 100% methanol.

[0047] Example 1

[0048] 1. Obtaining Aeroto-AUH-df6, an Aerotolerant Blautiae strain

[0049] Strictly anaerobic Blautiae AUH-JLD56 (CGMCC NO.8032) grown to the exponential growth phase after being cultured in an anaerobic workstation for 18 hours to 24 hours is inoculated into an acclimation medium at a 10% inoculation amount, and cultured in a common biochemical incubator. After being transferred 5 to 10 times under the condition of air oxygen, the ascorbic acid in the culture medium is successively reduced from 0.15% (reduced by 0.01% to 0.02% each time), the L-cysteine ​​is successively reduced from 0.15% (reduced by 0.01% to 0.02% each time), and the agar is successively reduced from 0.08% (reduced by 0.005% to 0.01% each time), and after the growth is stabilized (transferred 5 to 10 times), the next gradient is transferred to continue acclimation, and the like is followed to gradually reduce the ascorbic acid, L-cysteine ​​and agar content in the culture medium to zero. During the transfer process, HPLC tracking determination is used to detect the conversion of the substrate arctiin. Finally, an oxygen-tolerant mutant strain was obtained, which could grow in the presence of air oxygen and convert arctigenin into 3′-demethylarcigenin. The strain was named as Aeroto-AUH-df6.

[0050] 2. Purification and Cultivation of Aeroto-AUH-df6

[0051] The domesticated aerobic-resistant Blautiae Aeroto-AUH-df6 with transformation function was inoculated into 4 mL of fresh BHI liquid culture medium at 10% inoculation rate, and cultured in the BHI liquid culture medium at 37°C for 8h to 12h. 400 μL of the bacterial solution was added to a cryopreservation tube containing 400 μL of sterilized 10% skimmed milk powder, mixed, and 2 mm thick sterilized liquid paraffin was added to the surface. The tube was then preserved in an ultra-low temperature refrigerator at -80°C, and the functional microbial strains preserved at low temperature were regularly subjected to rejuvenation culture and transformation activity determination.

[0052] 3. Morphological observation and physiological and biochemical identification

[0053] The colony diameter of strain Aeroto-AUH-df6 in BHI solid culture medium is 6mm~7mm, milky white, with irregular colony edges, slightly white in the center but without obvious protrusions, smooth and moist; in BHI liquid culture medium, the bacteria are short rods of 2~3 bacteria connected together. Careful observation will reveal that there are partitions between the bacteria. Compared with the strict anaerobic Blautiae AUH-JLD56 before domestication, the aerobic mutant strain Aeroto-AUH-df6 obtained after domestication is significantly smaller, but there is no adhesion between the bacteria.

[0054] The strain was Gram-positive; the strain was urease-positive and could utilize most carbohydrates, such as sucrose, isoflavone, xylose, arabinose, glycerol, cellobiose, mannose, melezitose, raffinose, sorbitol, rhamnose, and trehalose; the strain was catalase-negative and indole-negative.

[0055] 4. Molecular Biological Identification

[0056] The total DNA of a single functional microbial strain was used as a template, and the universal primers 27F / 1492R (27F: 5′-AGAGTTTGATCCTGGCTCAG-3′; 1492R: 5′-GGTTACCTTGTTACGACT T-3′) were used as primers to amplify the 16S rDNA sequence by PCR. The PCR amplification reaction procedure was as follows: 94°C pre-deformation for 5 min; 94°C deformation for 1 min, 55°C annealing for 30 s, 72°C extension for 1.5 min, 29 cycles; 72°C termination extension for 10 min. The PCR amplification product was sent to Shanghai Bioengineering Co., Ltd. for DNA sequencing. The amplification results of 16S rDNA were as follows:

[0057]

[0058] The sequencing results were compared with other strains in the GenBank database by BLAST, and the 16S rDNA sequences of the standard strains of similar Blautia and Ruminococcus were obtained. The evolutionary distance between strain Aeroto-AUH-df6 and related closely related strains was obtained, and a phylogenetic tree was constructed, such as Figure 1 shown.

[0059] The strain Aeroto-AUH-df6 had the highest similarity with the strain Blautia sp. strain AUH-JL D56 (KF374935), with a 16S rDNA sequence similarity of 99.43% and a similarity of 99.01% with the Blautia wexlerae strain WAL 14507 (EF 036467). Therefore, the aerobic strain was preliminarily identified as a Blautia strain and named Aeroto-AUH-df6.

[0060] The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, abbreviated as CGMCC, on April 10, 2024. The strain deposit number is CGMCC NO.30297. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0061] Example 2

[0062] Preparation of pure arctigenin:

[0063] (1) The commercially available burdock seeds were crushed with a grinder, 10 g of the crushed burdock powder was weighed and added to a 250 mL conical flask, and 80 mL of a 90% methanol aqueous solution (solid-to-liquid ratio of 1:8) was added at the same time, and ultrasonic extraction was performed at room temperature for 15 min to obtain a burdock seed extract.

[0064] (2) Preparation method of crude arctigenin

[0065] After filtering the burdock fruit extract with filter paper, add commercial concentrated hydrochloric acid in a volume ratio of 1:4 to the extract to the filtrate, and keep it in a water bath at 80°C for 2 hours for acid hydrolysis. After the hydrolysis is completed, take it out, wait for the liquid temperature in the conical flask to drop to room temperature, add sodium hydroxide solution for neutralization, then add an equal volume of ethyl acetate solution for extraction, and evaporate the extract to dryness to obtain crude arctigenin.

[0066] The extraction effect was tested by high performance liquid chromatography. The testing conditions were: the chromatographic column was Kromasil C 18Reverse phase chromatographic column (4.6 mm × 250 mm, 5 μm), mobile phase A is a mixed solution of acetonitrile and 0.1% acetic acid aqueous solution in a volume ratio of 10:90, mobile phase B is a mixed solution of acetonitrile and 0.1% acetic acid aqueous solution in a volume ratio of 90:10, isocratic elution, mobile phase A: mobile phase B = 60:40 (v / v), flow rate: 1 mL / min, injection volume: 20 μL, detection wavelength: 280 nm.

[0067] Under the same mobile phase and detection conditions, the retention time of the acid hydrolysis product of the methanol aqueous solution extract of burdock fruit was completely consistent with that of arctigenin standard. Therefore, the acid hydrolysis product of the methanol aqueous solution extract of burdock fruit was identified as arctigenin.

[0068] (3) Preparation method of pure arctigenin

[0069] In order to prepare a large amount of the acid hydrolyzate of the methanol aqueous solution extract of Arctium lappa, the hydrolyzate peak was collected on HPLC using a preparative column. The separation conditions were: Kromasil C 18 The column was prepared (10.0 mm × 250 mm, 5 μm), the mobile phase A was a mixed solution of acetonitrile and 0.1% acetic acid aqueous solution in a volume ratio of 10:90, and the mobile phase B was a mixed solution of acetonitrile and 0.1% acetic acid aqueous solution in a volume ratio of 90:10; isocratic elution, mobile phase A: mobile phase B = 60:40 (v / v); flow rate: 2.0 mL / min; injection volume: 120 μL; detection wavelength: 280 nm. The collected solution was evaporated to dryness using a rotary evaporator, and the rotary evaporation temperature was 60°C.

[0070] Example 3

[0071] Application of Aeroto-AUH-df6, an Aerotolerant Blautiae strain, in Aerobic Transformation of Arctigenin

[0072] (1) Cultivation of Aeroto-AUH-df6

[0073] The low-temperature frozen aerobic Blautiae Aeroto-AUH-df6 was thawed and inoculated into a test tube containing fresh BHI liquid culture medium at a 15% inoculation rate, and activated and cultured at 37°C for 24 hours. The aerobic Blautiae Aeroto-AUH-df6 in the test tube was then transferred back into fresh BHI liquid culture medium at a 10% inoculation rate and cultured for 15 hours to obtain a seed solution.

[0074] (2) Co-culture of the substrate arctigenin with the aerotolerant Blautiae Aeroto-AUH-df6

[0075] The seed liquid of aerotolerant Blautiae Aeroto-AUH-df6 is inoculated into a BHI liquid culture medium at an inoculation rate of 10%, and 1.0 mL of a 200 mmol / L arctiin mother solution is first added to 200 mL of the BHI liquid culture medium to make the initial concentration of arctiin in the culture medium 1.0 mmol / L. After culturing in a constant temperature incubator for 8 hours, the arctiin mother solution is supplemented, and the addition is performed 4 times in total according to the above method to make the total concentration of arctiin in the culture medium 5 mmol / L. Finally, the substrate arctiin can be efficiently converted into 3′-demethyl arctiin by co-culturing in a constant temperature incubator for 3 days.

[0076] (3) Isolation, purification and identification of metabolites

[0077] The culture in the test tube was extracted twice with an equal volume of ethyl acetate, the extracts were combined, filtered and evaporated to dryness on a rotary evaporator, 100% chromatographic grade methanol was added for dissolution, and after passing through a 0.45 μm organic membrane, separation was performed on a semi-preparative high performance liquid chromatography, and the metabolites were collected in a clean conical flask and evaporated to dryness by rotary evaporation to obtain 3′-demethyl arctiin. The conditions for semi-preparative high performance liquid chromatography separation and purification were the same as those in Example 2 under the preparation method of pure arctiin. The chromatogram is shown in FIG. Figure 2 The UV absorption spectrum is shown in Figure 3 As shown in the mass spectrum Figure 4 shown.

[0078] The results of high performance liquid chromatography showed that the transformation product to be structurally identified had the same chromatographic peak retention time as the standard 3′-demethyl arctigenin, wherein the 3′-demethyl arctigenin standard was a transformation product generated by the inventors' team using strictly anaerobic Blautiae AUH-JLD56 to transform the substrate arctigenin in an anaerobic workstation, and the product was structurally identified by mass spectrometry and nuclear magnetic resonance hydrogen and carbon spectra. The molecular weight of the transformation product was found to be 358 by cation mass spectrometry, and its nuclear magnetic resonance hydrogen and carbon spectra data are as follows:

[0079] Proton spectrum ( 1 H-NMR) and carbon spectra ( 13 C-NMR) analysis, the spectrum analysis results are as follows:

[0080] 1H NMR(CDCl3,400MHz,): δ2.46-2.64(4H,m,H-2,3,7″),2.85(2H,d,J=5.5Hz,H-7′),3.81-3.84 (6H,s,-OCH3*2),3.88(1H,dd,J=8.54,7.6Hz,H-4),4.13(1H,dd,J=8.54,7.8Hz,H-4),6.48( 1H,d,J=1.94Hz,H-2″),6.51(1H,dd,J=8.19,1.94Hz,H-6′),6.57(1H,dd,J=8.19,1.94Hz,H- 6″), 6.66(1H,d,J=1.94Hz,H-2′), 6.75(1H,d,J=8.19Hz,H-5′), 6.77(1H,d,J=8.19Hz,H-5″).

[0081] 13 C NMR (CDCl3, 400MHz): δ179.9 (C-1), 149.0 (C-3″), 147.8 (C-4″), 144.0 (C-3′), 142.9 (C-4′), 130.0 (C-1″), 129 .9(C-1′),121.7(C-6′),120.7(C-6″),116.1(C-2′),115.2(C-5′),111.8(C-2″),111.4(C-5″),71.8(C-4),-Me 55.9,-Me55.8,46.6(C-2),40.9(C-3),38.2(C-7″),34.0(C-7′).

[0082] The experimental results of this study showed that the peak at the retention time of 8.7min in the HPLC was the conversion product peak, and the conversion product peak had exactly the same HPLC retention time as the 3′-demethylarcitogenin standard ( Figure 2 ). From the UV absorption spectrum, the conversion product has maximum UV absorption at 227nm and 280nm, which is completely consistent with the 3′-demethylarcigenin standard ( Figure 3 To identify the molecular weight of the conversion product, the purified conversion product was subjected to negative ion mass spectrometry analysis (ESI-MS). The mass spectrometry analysis results were: ESI (-): m / z 357 ([M+H] － ), according to the mass spectrometry analysis results, the molecular weight of the product peak of the substrate arctiin transformed by Aeroto-AUH-df6 should be 358, which is exactly the same as the molecular weight of the standard 3′-demethyl arctiin (C 20 H 22Therefore, the product peak of the substrate arctigenin transformed by Aeroto-AUH-df6 was identified as 3′-demethylarcigenin.

[0083] Dissolve 3′-demethyl arctigenin in 100% methanol and measure the optical rotation of the obtained product 3′-demethyl arctigenin at 25°C using a polarimeter, i.e., [α] 25 D is -57.4°(c=0.54×10 -3 ,MeOH).

[0084] Example 4

[0085] Transformation dynamics of arctigenin by aerotolerant Blautiae Aeroto-AUH-df6

[0086] In order to understand the speed of arctigenin conversion by Aeroto-AUH-df6, so as to determine the optimal conversion time, the conversion dynamics of arctigenin conversion by Aeroto-AUH-df6 were determined. The specific method is as follows:

[0087] The seed solution (the culture method is the same as in Example 1) cultured in advance was inoculated into a 250mL infusion bottle containing 100mL fresh BHI liquid medium according to a 10% inoculation amount, and 1.25mL of 40mmol / L arctigenin mother solution was added at the same time. After culturing in ordinary biochemical culture for 0h, 4h, 6h, 8h, and 12h, 100μL of the culture was taken, extracted with 500μL ethyl acetate, and 400μL of the extract was taken. After evaporating the extract to dryness, 80μL methanol was added, and the substrate was detected by HPLC after filtering. The transformation dynamics of the strain were drawn according to the concentrations of the substrate and the product at different culture times. The results are shown in Table 1 and Figure 5 shown.

[0088] Table 1 Substrate and product concentrations at different culture times (mmol / L)

[0089]

[0090] Depend on Figure 5 It can be seen that the product 3'-demethylarcigenin began to be generated 4 hours after the addition of the substrate arctigenin. After 8 hours of continuous cultivation, the added substrate arctigenin was completely converted into 3'-demethylarcigenin, and the product amount basically stopped increasing from 8 hours to 12 hours.

[0091] Example 5

[0092] Determination of the maximum conversion capacity of the aerotolerant strain Aeroto-AUH-df6 to arctigenin at different concentrations

[0093] The seed solution cultured in advance (the culture method is the same as in Example 1) was inoculated into fresh BHI liquid culture medium at a 10% inoculation rate, and different concentrations of arctigenin mother solution were added during the culture period, and the culture was continued to be co-cultured in a common biochemical incubator for 3 days, and then the culture was extracted with an equal volume of ethyl acetate, and the conversion rate was calculated according to the standard curve and the peak area (conversion rate = product concentration / (remaining substrate concentration+product concentration)×100%). The results are as follows: Figure 6 And as shown in Table 2.

[0094] Table 2 Conversion rate at different substrate concentrations in culture medium

[0095]

[0096]

[0097] The results showed that the average conversion rates of arctiin with initial concentrations of 4.6mmol / L and 5.8mmol / L in the culture medium by aerotolerant Blautiae Aeroto-AUH-df6 were 99.6% and 99.1%, respectively; when the substrate concentration increased to 8.2mmol / L, the conversion efficiency of arctiin by aerotolerant Blautiae Aeroto-AUH-df6 decreased, and the average conversion rate dropped to 90.0%; when the substrate concentration increased to 9.4mmol / L, the conversion ability of arctiin by aerotolerant Blautiae Aeroto-AUH-df6 decreased significantly, and the average conversion rate dropped to 80.7%.

[0098] In summary, the aerotolerant Blautiae Aeroto-AUH-df6 with a preservation number of CGMCC NO.30297 provided by the present invention can efficiently convert the substrate arctiin into the product 3′-demethyl arctiin under the condition of air oxygen, and the conversion performance is stable, and a simple method is provided for the conversion and synthesis of 3′-demethyl arctiin, which solves the problem that 3′-demethyl arctiin cannot be mass-produced under the condition of air oxygen in the prior art, and has a great promoting effect on the pharmacological activity research of 3′-demethyl arctiin and the development of new drugs.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An aerotolerant Blautia sp. Aeroto-AUH-df6, characterized in that: Its deposit number is CGMCC NO.30297.

2. Use of the aerotolerant Blautiae Aeroto-AUH-df6 according to claim 1 in aerobic conversion of arctiin to synthesize 3'-demethylarcitinin.

3. A method for synthesizing 3'-demethyl arctigenin, characterized in that: Arctigenin is used as a substrate and the culture of the aerotolerant Blautiae Aeroto-AUH-df6 according to claim 1 is used for transformation under the condition of air oxygen to obtain 3'-demethylarcigenin.

4. The method for synthesizing 3'-demethyl arctigenin according to claim 3, characterized in that: The specific steps include: The seed liquid of aerotolerant Blautiae Aeroto-AUH-df6 is inoculated into a culture medium, arctiin mother liquid is added, the culture is carried out at a constant temperature under air oxygen conditions, and 3'-demethyl arctiin is obtained by separation and purification.

5. The method for synthesizing 3'-demethyl arctigenin according to claim 4, characterized in that: The inoculation amount of the seed solution of the aerotolerant Blautiae Aeroto-AUH-df6 is 5% to 10% (v / v).

6. The method for synthesizing 3'-demethyl arctigenin according to claim 4, characterized in that: The concentration of the arctigenin mother solution is 200 mmol / L to 250 mmol / L.

7. The method for synthesizing 3'-demethyl arctigenin according to claim 4, characterized in that: The final concentration of arctigenin in the culture medium is 5 mmol / L to 10 mmol / L; and / or The constant temperature culture temperature is 36° C. to 38° C., and the constant temperature culture time is 2 days to 3 days.

8. The method for synthesizing 3'-demethyl arctigenin according to claim 4, characterized in that: The separation and purification specifically comprises the following steps: adding ethyl acetate to the culture for extraction, filtering the extract and then evaporating to dryness, adding methanol to dissolve, filtering and then performing semi-preparative high performance liquid chromatography separation, collecting the eluate with the product peak, evaporating to dryness, and obtaining a pure product of 3'-demethylarcitogenin.

9. The method for synthesizing 3'-demethyl arctigenin according to claim 8, characterized in that: The conditions for the semi-preparative HPLC separation are: Chromatographic column: Kromasil C 18 Preparation column, 250mm×10mm, 5μm; mobile phase A: a mixed solution of acetonitrile and acetic acid aqueous solution in a volume ratio of 8-12:92-88, mobile phase B: a mixed solution of acetic acid aqueous solution and acetonitrile in a volume ratio of 8-12:92-88, and the volume concentration of acetic acid aqueous solution is 0.08%-0.12%; isocratic elution, mobile phase A: mobile phase B = 58-62:42-38 (v / v); flow rate: 1.8mL / min-2.2mL / min; injection volume: 80μL-150μL; detection wavelength: 278nm-282nm.

10. The method for synthesizing 3'-demethyl arctigenin according to claim 4, characterized in that: The preparation method of the seed liquid of the oxygen-resistant Blautiae Aeroto-AUH-df6 comprises the following steps: thawing the frozen and stored oxygen-resistant Blautiae Aeroto-AUH-df6, inoculating it into a BHI liquid culture medium at an inoculation amount of 15% to 20% (v / v), and culturing it at 36° C. to 38° C. for 20 h to 25 h to obtain the activation liquid of the oxygen-resistant Blautiae Aeroto-AUH-df6; inoculating the activation liquid into a BHI liquid culture medium at an inoculation amount of 5% to 10% (v / v), and culturing it at 36° C. to 38° C. for 12 h to 18 h to obtain the seed liquid of the oxygen-resistant Blautiae Aeroto-AUH-df6.

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