CN103981104B - Endophytic fungus and method for biologically converting glycyrrhizic acid into glycyrrhetinic glycoside by using same - Google Patents
Endophytic fungus and method for biologically converting glycyrrhizic acid into glycyrrhetinic glycoside by using same Download PDFInfo
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- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 title claims abstract description 70
- VTAJIXDZFCRWBR-UHFFFAOYSA-N Licoricesaponin B2 Natural products C1C(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2)C(O)=O)C)(C)CC2)(C)C2C(C)(C)CC1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O VTAJIXDZFCRWBR-UHFFFAOYSA-N 0.000 title claims abstract description 57
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 239000001685 glycyrrhizic acid Substances 0.000 title claims abstract description 57
- 229960004949 glycyrrhizic acid Drugs 0.000 title claims abstract description 57
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 title claims abstract description 57
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- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to an endophytic fungus and a method for biologically converting glycyrrhizic acid into glycyrrhetinic glycoside, wherein the fungus is named as DX-SES3 and is identified as < i > Microphaeropsis </i > < i >? sarundini, and has been deposited in China center for type culture Collection with a deposition number of CCTCC? M? 2014177. The strain can generate beta-glucuronidase under the induction of glycyrrhizic acid, and further catalyze glycyrrhizic acid to produce GAMG; the GAMG produced is enriched on the surface of the thallus, 75% ethanol is used for washing the thallus, and the GAMG crude product is obtained after decompression, concentration and spray drying, and the purity is 89.4% by HPLC detection. The invention adopts a biotransformation method, which not only has high glycyrrhizic acid conversion rate and is green, environment-friendly and pollution-free, but also has GAMG enriched on the surface of the thallus, and the product separation and purification are simple and easy.
Description
Technical field
The present invention relates to conversion technology field and product separating technique, relating generally to a strain can be single by glycyrrhizic acid,Directed the Dongxiang Wild Rice endogenetic fungus that is converted into liquorice enoxolone (GAMG) and GAMG preparation method for separating and purifying.
Technical background
Glycyrrhizic acid (GL) is one of main active in Radix Glycyrrhizae, is the highest triterpene compound of content in Radix Glycyrrhizae,Structure as shown in Figure 1, connects two glucuronic acids by pentacyclic triterpene saponin by glycosidic bond and forms. Modern pharmacology grindsStudy carefully and show, glycyrrhizic acid has anti-inflammatory, antiviral, antiallergy, the pharmacological action such as antitumor, or a kind of high sugariness, low in caloriesNovel sweetener, there is certain using value.
The glucuronic acid base that glycyrrhizic acid is removed its end through beta-glucuronidase enzyme hydrolysis just generate GAMG(asFig. 1), be an important derivative of glycyrrhizic acid. Liquorice enoxolone is again GAMG (GAMG), and it shouldWith being worth much larger than glycyrrhizic acid. First, application aspect in food, the sugariness of GAMC is about 941 times of sucrose sweetness, is Radix GlycyrrhizaeMore than 5 times of sour-sweet degree, GAMG not only has stronger continuation sweet taste, is a kind of high sugariness, novel sweetener low in calories, andCan effectively reduce because of diseases such as obesity that the absorption of high heat sweetener causes, diabetes, hyperlipemia, carious teeth; Secondly, at medicineThe application of object space face, GAMG and glycyrrhizic acid have similar pharmacological action, but liquorice enoxolone middle polarity, in vivo solubilityPreferably and easier transmembrane transport, better than glycyrrhizic acid bioavilability, its bioavilability is better than Radix Glycyrrhizae acids medicine and sweetGrass time acids medicine, has significant novel drugs exploitation and is worth, as the Epstein-Barr virus EA to TPA inductionThe inhibiting rate demonstration forming, the inhibiting rate of GAMG has reached 100%, and the inhibiting rate of glycyrrhizic acid only has 84.4%, many research tablesUnderstand that there is good effect the aspects such as antitumor, antiviral, anti-inflammatory, and confirm that its multiple pharmacologically active is not second to Radix GlycyrrhizaeAcid, the most important is that liquorice enoxolone is more safer than glycyrrhizic acid, the LD50 of liquorice enoxolone is 5000mg/kg, and glycyrrhizic acidLD50 is 805mg/kg; Finally, GAMG also has important application in cosmetics, and GAMG can make oiliness perfume or hormone increaseMolten, can be used in transparent dressing product, can not produce foam, also can be used in face cream or emulsion, be mixed with stable oil-in-water breastLiquid etc. Therefore produce and develop GAMG and there is very important using value and realistic meaning.
The acquiring way of GAMG is fewer, and main path has following several: the one, and chemical synthesis is synthesized GAMG,Brieskorn etc. and Mizutani etc. have synthesized GAMG first, but synthetic route is long, and yield is very low. The 2nd, traditional chemical waterSolution, the glucuronic acid base that removes glycyrrhizic acid by hydrolysis generates liquorice enoxolone, but this method is to two of glycyrrhizic acid glycosidic bondsSelectively low, can not directionally hydrolyzing generate liquorice enoxolone, accessory substance is many, and yield is lower, has highly energy-consuming simultaneously, the lacking of high pollutionPoint. The 3rd, the β-D-Glucose aldehydic acid enzyme hydrolysis glycyrrhizic acid that utilizes Institute of Micro-biology to produce prepares, and this is also currentThe main acquisition approach of GAMG, and its β-D-Glucose aldehydic acid enzyme dosage is excessive, source inconvenience is also uneconomical, and product isDifficult separation and purification. In addition, according to the legislation of European Union and the U.S., the product obtaining by chemical method is not natural materials, applicationBe restricted in field of food, and the material that utilizes enzyme process or microbial method (being biotransformation method) to obtain is considered to naturalMaterial. Bio-transformation refers to and utilizes the enzyme of microorganism, animals and plants and cultivating system or its generation to carry out structure to xenobionticsThe biochemical reaction process of modifying, its essence is to utilize the enzyme that living things system produces that xenobiontics is carried out to enzymatic is anti-Should. Utilize bio-transformation glycyrrhizic acid to generate GAMG and had been reported, it not only has advantages of that selectivity is high, efficiency is high, andMethod is simple, be convenient to separate, and is the desirable approach that will obtain from now on GAMG.
The inventor separates and screens from the tissue of Chinese Second Class Key Protected Plant grass Dongxiang Wild Rice healthTo can be by the endogenetic fungus that is converted into GAMG of the single orientation of glycyrrhizic acid---bacterial strain DX-SES3 (MicrosphaeropsiSarundinis). This bacterium has been preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCCM2014177. This bacterium energyUnder the induction of glycyrrhizic acid, glycyrrhizic acid orientation can be converted into GAMG, and conversion ratio is higher, particularly points out, syntheticGAMG richness is amassed on the surface of bacterium ball, is beneficial to greatly the separation and purification work of follow-up GAMG, and this is for utilizing this bacterial strain bio-transformationGenerate preparation GAMG new Research Thinking is provided.
Summary of the invention
The object of this invention is to provide a strain and there is the directed Dongxiang Wild Rice endogenetic fungus DX-that glycyrrhizic acid is GAMG that transformsSES3 (Microsphaeropsisarundinis). It is poor that the present invention has not only overcome in existing technology catalysis directionality,Contaminated environment, highly energy-consuming, the shortcoming that low conversion rate etc. are not enough, but also avoided the complicated procedures of forming of GAMG separation and purification.
The endogenetic fungal bacterial strain that transfer glycyrrhizic acid of the present invention is GAMG, called after DX-SES3, is categorized as small spherical shell spore and belongs toBacterium Microsphaeropsisarundinis, has been preserved in Chinese Typical Representative culture collection center, and the preservation time is 2014 5The moon 3, preserving number is CCTCCM2014177.
The morphological feature of Dongxiang endogenetic fungal bacterial strain DX-SES3 of the present invention is: on PDA, cultivate 5-7d for 28 DEG C,5 days colony diameters are 20mm, and 7 days colony diameters are 30-35mm, and within 7 days, colony diameter is unchanged; Quality is cotton-shaped, central protrusion,Surface canescence, middle color depth, edge color is shallow, old rear color burn, without penetrating fluid, without soluble pigment, bacterium colony reverse side is blackLook; Mycelia elongate curved, is black or grey black; The spore of attending to anything else is less, is pupa worm shape (as Fig. 2).
Dongxiang Wild Rice endogenetic fungus DX-SES3 of the present invention (Microsphaeropsisarundinis) baseBecause accession number is KC871028.
Dongxiang Wild Rice endogenetic fungus DX-SES3 of the present invention (Microsphaeropsisarundinis) turnsThe directed liquorice enoxolone that generates of glycyrrhizic acid comprises the following steps:
Step 1, by Dongxiang Wild Rice endogenetic fungus DX-SES3 (Microsphaeropsisarundinis), inoculationIn PDA slant medium, cultivate activation 72~84 hours, and make spore suspension, be then seeded in seed culture medium,28 ± 1 DEG C, 150~300r/min, shaking table is cultured to exponential phase.
Step 2, according to the inoculum concentration of mass fraction 10%~30%, the seed liquor of above-mentioned exponential phase is seeded to and is containedIn the conversion culture medium of glycyrrhizic acid, transform and produce liquorice enoxolone to content substantially constant.
The GAMG on step 3, flushing bacterium ball surface, and separation and purification GAMG.
Preferably, described DX-SES3 (Microsphaeropsisarundinis) transforms the directed generation of glycyrrhizic acidGAMG, the consisting of of seed culture medium in step 1: in every liter of culture medium containing glucose 10~40g, glycyrrhizic acid (or ammonium glycyrrhetateSalt) 0.1~1g, KH2PO41.0~3.0g,NH4NO32.0~5.0g, NaCl0.3~0.8g, dusty yeast 0.05~0.3g,MgSO40.1~0.5g,CaCl20.01~0.5g,FeSO40.014g,ZnSO4·7H2O2.9mg,MnCl2·4H2O2.0mg,CuSO4·5H2O0.25mg,CoCl2·6H2O0.24mg,Na2MoO4·2H2O0.24mg,H3BO30.03mg, all the other are pure water, adjusting pH is 5.0~7.0.
Preferably, described DX-SES3 (Microsphaeropsisarundinis) transforms the directed generation of glycyrrhizic acidGAMG transforms culture medium and consists of in step 2: in every liter of culture medium, contain glycyrrhizic acid (or ammonium glycyrrhizunate) 2~30g,KH2PO41.0~3.0g,NH4NO32.0~4.0g, NaCl0.3~0.8g, dusty yeast 0.05~0.1g,MgSO40.1~0.5g,CaCl20.01~0.5g,FeSO40.014g,ZnSO4·7H2O2.9mg,MnCl2·4H2O2.0mg,CuSO4·5H2O0.25mg,CoCl2·6H2O0.24mg,Na2MoO4·2H2O0.24mg,H3BO30.03mg, all the other are pure water, adjusting pH is 3.5~9.0. Wherein glycyrrhizic acid (or ammonium glycyrrhizunate) be thalline produce β-The derivant of glucuronidase.
Preferably, described DX-SES3 (Microsphaeropsisarundinis) transforms the directed generation of glycyrrhizic acidGAMG, in step 2, condition of culture is: 28 DEG C~60 DEG C, rotating speed 150~300r/min.
Preferably, described DX-SES3 (Microsphaeropsisarundinis) transforms the directed generation of glycyrrhizic acidGAMG, in step 3, the separation and purification of liquorice enoxolone comprises following steps:
Buchner funnel suction filtration or six layers of filtered through gauze, collect thalline, and the alcohol flushing thalline with 75% 3-5 time is collected ethanolSolution, rear reduced pressure concentration, then spraying is dry, obtains the higher thick finished product of GAMG of purity.
Last the present invention adopts high performance liquid chromatography and LC-MS further to detect GAMG, confirms glycyrrhizic acid conversion ratio, Radix GlycyrrhizaeThe purity of inferior glycosides and yield.
Brief description of the drawings
Fig. 1 is Dongxiang Wild Rice endogenetic fungus DX-SES3 of the present invention (Microsphaeropsisarundinis)Colonial morphology and ascus form, in figure, A:PDA colonial morphology; B:CA colonial morphology; C: pupa worm shape sporangium (microscopeLower 40 ×);
Fig. 2 glycyrrhizic acid is generated GAMG by beta-glucuronidase enzyme hydrolysis;
Fig. 3 is Dongxiang Wild Rice endogenetic fungus DX-SES3 of the present invention (Microsphaeropsisarundinis)The HPLC that transforms glycyrrhizic acid product detects;
Fig. 4 endogenetic fungus DX-SES3 (Microsphaeropsisarundinis) transforms the LC-MS of glycyrrhizic acid productDetect.
Specific embodiments
Below in conjunction with example, the present invention is described in detail
Embodiment 1: Dongxiang Wild Rice bacterial strain DX-SES3 (Microsphaeropsisarundinis) in the present inventionSeparate
(1) gather healthy Dongxiang Wild Rice plant sample, take back in experiment 12h and process.
(2) plant that selects wild rice material to grow fine without illness is rinsed 1-2h under running water, the withered jaundice of removalBranches and leaves coring, after cleaning, chooses healthy root, stem and leaf, and it is some that root, stem, leaf are cut into the segment of 3cm.
(3) be then placed in the 1-2%NaClO solution of new preparation, stem and leaf soak 5min, and root is processed 4min, then useNewly 2.5% of preparation Na2S2O3Soak 10min, sterile water wash 3 times, then 75% of the new preparation of use alcohol immersion 1Min, sterile water wash 3 times.
(4) the above-mentioned stem of handling well is cut two with the scissors of sterilizing, then will be removed the stem vertical profile at two, one pointBe two, then be cut into the fritter of 0.1 × 0.5cm. The edge of leaf is cut off with sterilized scissors, the leaf that has cut off edgeSheet is also cut into the approximately fritter of 0.2 × 0.5cm, and plant tissue fritter after treatment is grown surely containing 50mgL-1The PDA of chloramphenicol,On MEA, CA culture medium, be placed in 28 DEG C of cultivations. In order to check surperficial sterilization effect, establish contrast and carry out the inspection of sterilization effect, useLiquid-transfering gun is drawn the last sterilized water cleaning of 200 μ L, is coated on uniformly on plating medium, in contrast.
(5) situation that grows that every day, endogenetic fungus was observed in timing, the fungi picking that incision is newly grown is received PDA and is cultivatedOn base, purifying is cultivated until obtain pure bacterial strain.
Embodiment 2: the present invention filters out the directed Dongxiang Wild Rice bacterial strain DX-SES3 that transforms glycyrrhizic acid generation GAMG(Microsphaeropsisarundinis)。
(1) dull and stereotyped primary dcreening operation: by the endogenetic fungus activation of the above-mentioned Dongxiang Wild Rice being separated to, be inoculated in glycyrrhizic acid as onlyIn the solid screening and culturing base of one carbon source, control group replaces glycyrrhizic acid as sole carbon source with glucose. 28 DEG C, cultivate 7d, sieveSelect and can give birth to well-grown bacterial strain.
Described screening and culturing base is; In every liter of culture medium, contain glycyrrhizic acid 3.0g, KH2PO42.2g,NH4NO33.0G, NaCl0.5g, dusty yeast 0.05g, MgSO40.12g,CaCl20.014g,FeSO40.014g,ZnSO4·7H2O2.9mg,MnCl2·4H2O2.0mg,CuSO4·5H2O0.25mg,CoCl2·6H2O0.24mg,Na2MoO4·2H2O0.24mg,H3BO30.03mg, agar powder 20g(fluid nutrient medium does not add), all the other are pure water, pH6.0.
(2) shaking flask is sieved again: above-mentioned dull and stereotyped primary dcreening operation inoculation out (is used to the glucose of 20g/L to seed culture mediumReplace the glycyrrhizic acid of the 3.0g/L in screening and culturing base) in 28 DEG C, 150rpm cultivate 2d, then press 20% inoculum concentrationBeing forwarded to glycyrrhizic acid is in the liquid screening culture medium of sole carbon source, and establishes two contrasts: the one, in liquid screening culture medium notWhether access thalline, decompose to detect glycyrrhizic acid in culture medium; The 2nd, bacterial strain is transferred to glucose replacement glycyrrhizic acid workIn screening and culturing base for sole carbon source, 28 DEG C, 150rpm shaking table cultivation 4d, collect zymotic fluid, with TLC, HPLC and LC-MS detects the product transforming.
Experimental result shows: Dongxiang Wild Rice endogenetic fungus DX-SES3 (Microsphaeropsisarundinis) energyOrientation is converted into liquorice enoxolone (as Fig. 3, Fig. 4), glycyrrhizic acid conversion ratio ((GL molar concentration after initial GL molar concentration-conversion)/Initial GL molar concentration × 100%) be 72.4%, GAMG productive rate (transform after GAMG molar concentration/initial GL molar concentration ×100%) be 67.3%.
Embodiment 3: Dongxiang Wild Rice bacterial strain DX-SES3 (Microsphaeropsisarundinis) transforms glycyrrhizic acidGenerate GAMG
Dongxiang Wild Rice bacterial strain DX-SES3 (Microsphaeropsisarundinis) after activation is linked into kind28 DEG C of sub-culture mediums, 250rpm cultivate 3d, are then forwarded to and transform in culture medium by 30% inoculum concentration, 35 DEG C, 250rpmShaking table is cultivated 7d. The centrifugal thalline that goes, collects zymotic fluid, detects converted product with HPLC.
Consisting of of described seed culture medium: contain glucose 25g, glycyrrhizic acid (or ammonium glycyrrhizunate) in every liter of culture medium0.2g,KH2PO42.0g,NH4NO33g, NaCl0.5g, dusty yeast 0.1g, MgSO40.12g,CaCl20.2g,FeSO40.014g,ZnSO4·7H2O2.9mg,MnCl2·4H2O2.0mg,CuSO4·5H2O0.25mg,CoCl2·6H2O0.24mg,Na2MoO4·2H2O0.24mg,H3BO30.03mg, all the other are pure water, adjust pH to be6.0。
Described conversion culture medium consists of: in every liter of culture medium, contain mono-ammonium glycyrrhizinate 5g, KH2PO42.2g,NH4NO33g, NaCl0.5g, dusty yeast 0.1g, MgSO40.12g,CaCl20.4g,FeSO40.014g,ZnSO4·7H2O2.9mg,MnCl2·4H2O2.0mg,CuSO4·5H2O0.25mg,CoCl2·6H2O0.24mg,Na2MoO4·2H2O0.24mg,H3BO30.03mg, all the other are pure water, adjusting pH is 7.0.
After conversion finishes, 8 layers of filtered through gauze or suction filtration thalline, then use 75% alcohol flushing thalline 3-5 time, collects secondAlcoholic solution, and detect converted product, testing result is that glycyrrhizic acid conversion ratio is that 80.5%, GAMG productive rate is 75.2%.
The ethanolic solution of gained is after reduced pressure concentration, then spraying is dry, obtains the thick finished product of GAMG, and detecting purity is 89.4%.
Embodiment 4: Dongxiang Wild Rice bacterial strain DX-SES3 (Microsphaeropsisarundinis) transforms glycyrrhizic acidGenerate the detection of GAMG
It is as follows that high performance liquid chromatography detects converted product step: converted product (zymotic fluid and methyl alcohol are washed the amalgamation liquid of bacterium liquid)With after 5 times of chromatogram methyl alcohol dilutions, with organic filter membrane, (aperture is that 0.22 μ m or 0.45 μ m) use high performance liquid chromatograph after filteringAnalyzing and testing. Chromatographic condition: instrument is that chromatographic column is that (250 × 4.6mm, m), detector is 4 μ J ' sphereODS-H80PDA, detects wavelength 251.1nm, and mobile phase is methyl alcohol: tri-distilled water (acetic acid is adjusted pH2.85)=81:19, and sample size: 20 μ L,Flow velocity is 1mL/min, column oven: 35 ° of C.
LC-MS (LC-MS) detects converted product: the processing method of sample is the same with HPLC method processing method. Detect ginsengNumber is: instrument: Agilent6120QuadrupoleLC/MS, chromatographic column: AgilentStableBondSB-C18(2.1×50mm, 1.8 μ m), detector: level Four bar mass detector, mobile phase: methyl alcohol: tri-distilled water=81:19, sample size: 5 μ L, streamSpeed: 0.2mL/min, column oven: 25 ° of C, ionization mode: EIS(-), select ion detection (SIM) mass-to-charge ratio (M/Z): 821.9With with 645.5, it is corresponding that to detect GL(molecular weight be 822.9) and GAMG(molecular weight be 646.81).
Annex: the ITS sequence of bacterial strain DX-SES3 (Microsphaeropsisarundinis)
TTCCGTAAGGTGAACCTGCGGAAGGATCATTACCAATTCAACGGTGTGGTCGCGGCCTCCGGGGGCTTCCCTCCGGGCGGTAGAGGTAACACTCTCACGCGCCACATGTCTGAATCCTTTTTTTTACGAGCACCTTTCGTTCTCCCTCGGTGGGGCAACCTGCCGTTGGAACTTATCAAAAACCTTTTTTGCATCTAGCATTACCTGTTCAGATACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCATGGGGCATGCCTGTTCGAGCGTCATCTACACCCTCAAGCTCTGCTTGGTGTTGGGCGTCTGTCCCGCCTCCGCGCGTGGACTCGCCCCAAATTCATTGGCAGCGGTCTTTGCCTCCTCTCGCGCAGCACATTGCGCTTCAGAGGGGCGTGGGCCGCGTCCACGAAGCAACATTACCGTCTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGG。
Annex: the ITS sequence of bacterial strain DX-SES3 (Microsphaeropsisarundinis)
TTCCGTAAGGTGAACCTGCGGAAGGATCATTACCAATTCAACGGTGTGGTCGCGGCCTCCGGGGGCTTCCCTCCGGGCGGTAGAGGTAACACTCTCACGCGCCACATGTCTGAATCCTTTTTTTTACGAGCACCTTTCGTTCTCCCTCGGTGGGGCAACCTGCCGTTGGAACTTATCAAAAACCTTTTTTGCATCTAGCATTACCTGTTCAGATACAAACAATCGTTACAACTTTCAACAATGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATTCCATGGGGCATGCCTGTTCGAGCGTCATCTACACCCTCAAGCTCTGCTTGGTGTTGGGCGTCTGTCCCGCCTCCGCGCGTGGACTCGCCCCAAATTCATTGGCAGCGGTCTTTGCCTCCTCTCGCGCAGCACATTGCGCTTCAGAGGGGCGTGGGCCGCGTCCACGAAGCAACATTACCGTCTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGG。
Claims (5)
1. the method that endogenetic fungus bio-transformation glycyrrhizic acid is GAMG, comprises the steps:
1) activation of endogenetic fungus and the preparation of seed liquor, described endogenetic fungus called after DX-SES3, is categorized as small spherical shell sporeBelong to bacterium Microsphaeropsisarundinis, be preserved in Chinese Typical Representative culture collection center, the preservation time is 2014In on May 3, in, preserving number is CCTCCM2014177;
2) GAMG bio-transformation: the seed liquor of step 1) is joined to conversion culture medium by the inoculum concentration of mass fraction 20-40%In, carry out the bio-transformation of GAMG;
3) separation of GAMG preparation: the GAMG richness after bio-transformation is gathered in the surface of endogenetic fungus bacterium ball, Buchner funnel suction filtration orSix layers of filtered through gauze, collect thalline, and the ethanolic solution that is 20%~100% by volume ratio rinses thalline 3-5 time, collect ethanol moltenLiquid, rear reduced pressure concentration, then spraying is dry, obtains the larger GAMG finished product of purity, and detect with HPLC, LC-MS.
2. the method that endogenetic fungus bio-transformation glycyrrhizic acid as claimed in claim 1 is GAMG, is characterized in that described kindSub-liquid is cultivated and is prepared as: by endogenetic fungus DX-SES3(Microsphaeropsisarundinis) be inoculated into PDA inclined-plane and cultivateIn base, cultivate activation 72~108 hours for 20~30 DEG C, and make spore suspension, spore suspension is joined to seed culture mediumIn, 20~30 DEG C, 150~300r/min, shaking table is cultured to exponential phase.
3. the method that endogenetic fungus bio-transformation glycyrrhizic acid as claimed in claim 1 is GAMG, is characterized in that described kindConsisting of of sub-culture medium: in every liter of culture medium, contain glucose 10~40g, glycyrrhizic acid or ammonium glycyrrhizunate 0.1~1g,KH2PO41.0~3.0g,NH4NO32.0~5.0g, NaCl0.3~0.8g, dusty yeast 0.05~0.3g, MgSO40.1~0.5g,CaCl20.01~0.5g,FeSO40.014g,ZnSO4·7H2O2.9mg,MnCl2·4H2O2.0mg,CuSO4·5H2O0.25mg,CoCl2·6H2O0.24mg,Na2MoO4·2H2O0.24mg,H3BO30.03mg, itsRemaining is pure water, and adjusting pH is 5.0~7.0.
4. the method that endogenetic fungus bio-transformation glycyrrhizic acid as claimed in claim 1 is GAMG, is characterized in that described turningChanging condition of culture is: 28 DEG C~40 DEG C, and rotating speed 150~280r/min.
5. the method that endogenetic fungus bio-transformation glycyrrhizic acid as claimed in claim 1 is GAMG, is characterized in that described turningChanging culture medium consists of: in every liter of culture medium, contain glycyrrhizic acid or ammonium glycyrrhizunate 2~30g, KH2PO41.0~3.0g,NH4NO32.0~4.0g, NaCl0.3~0.8g, dusty yeast 0.05~0.1g, MgSO40.1~0.5g,CaCl20.01~0.5g,FeSO40.014g,ZnSO4·7H2O2.9mg,MnCl2·4H2O2.0mgCuSO4·5H2O0.25mg,CoCl2·6H2O0.24mg,Na2Mo4·2H2O0.24mg,H3BO30.03mg, all the other are pure water, adjustPH is 3.5~9.0, and wherein glycyrrhizic acid or ammonium glycyrrhizunate are the derivants that thalline produces beta-glucuronidase enzyme.
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