CN103992953B - Dongxiang wild rice endophytic fungus for converting glycyrrhizic acid to generate glycyrrhetinic acid glycoside - Google Patents
Dongxiang wild rice endophytic fungus for converting glycyrrhizic acid to generate glycyrrhetinic acid glycoside Download PDFInfo
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- enoxolone
- liquorice
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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 106
- 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 58
- 229960004949 glycyrrhizic acid Drugs 0.000 title claims abstract description 58
- 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 55
- 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 55
- 239000001685 glycyrrhizic acid Substances 0.000 title claims abstract description 55
- 235000019410 glycyrrhizin Nutrition 0.000 title claims abstract description 55
- 241000233866 Fungi Species 0.000 title claims abstract description 16
- 241000746966 Zizania Species 0.000 title abstract 2
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 38
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- 244000005700 microbiome Species 0.000 claims abstract description 4
- MPDGHEJMBKOTSU-YKLVYJNSSA-N 18beta-glycyrrhetic acid Chemical compound C([C@H]1C2=CC(=O)[C@H]34)[C@@](C)(C(O)=O)CC[C@]1(C)CC[C@@]2(C)[C@]4(C)CC[C@@H]1[C@]3(C)CC[C@H](O)C1(C)C MPDGHEJMBKOTSU-YKLVYJNSSA-N 0.000 claims description 48
- MPDGHEJMBKOTSU-UHFFFAOYSA-N Glycyrrhetinsaeure Natural products C12C(=O)C=C3C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C1(C)CCC1C2(C)CCC(O)C1(C)C MPDGHEJMBKOTSU-UHFFFAOYSA-N 0.000 claims description 48
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- WNIFXKPDILJURQ-UHFFFAOYSA-N stearyl glycyrrhizinate Natural products C1CC(O)C(C)(C)C2CCC3(C)C4(C)CCC5(C)CCC(C(=O)OCCCCCCCCCCCCCCCCCC)(C)CC5C4=CC(=O)C3C21C WNIFXKPDILJURQ-UHFFFAOYSA-N 0.000 claims description 48
- 241000228197 Aspergillus flavus Species 0.000 claims description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 17
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- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 8
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 7
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 7
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 7
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 7
- 239000011565 manganese chloride Substances 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000011684 sodium molybdate Substances 0.000 claims description 7
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 7
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 7
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- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 5
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims 2
- 102000005348 Neuraminidase Human genes 0.000 claims 1
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- 241001597008 Nomeidae Species 0.000 claims 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims 1
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- 239000000741 silica gel Substances 0.000 claims 1
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- 102000053187 Glucuronidase Human genes 0.000 abstract description 6
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- ILRKKHJEINIICQ-OOFFSTKBSA-N Monoammonium glycyrrhizinate Chemical compound N.O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@H]1CC[C@]2(C)[C@H]3C(=O)C=C4[C@@H]5C[C@](C)(CC[C@@]5(CC[C@@]4(C)[C@]3(C)CC[C@H]2C1(C)C)C)C(O)=O)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O ILRKKHJEINIICQ-OOFFSTKBSA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Dongxiang wild rice endophytic fungus for converting glycyrrhizic acid to generate glycyrrhetinic acid glycoside, and is identified and named asAspergillus flavusDX-SEL 2. Has been preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2013686. The strain can induce the production of beta-glucuronidase in a conversion culture medium with glycyrrhizic acid as an inducer, and then hydrolyze to remove a glucuronide group at the tail end of a glycyrrhizic acid molecule to generate the glycyrrhetinic acid glycoside (secreted in a fermentation liquid). Further separating and purifying by organic solvent extraction, normal phase silica gel column chromatography, reverse phase column chromatography, etc. to obtain glycyrrhetinic glycoside. The invention adopts the microorganism catalytic conversion, and has the advantages of environmental protection, no pollution, simple conversion step, single product, high conversion rate and the like.
Description
Technical field
The present invention relates to technical field of biotransformation, particularly relates to a strain conversion glycyrrhizic acid and generates the Dongxiang open country of liquorice enoxolone
Raw rice endogenetic fungus.
Background technology
Glycyrrhizic acid (GL) is one of main active in Radix Glycyrrhizae.The structure of glycyrrhizic acid such as Fig. 3, is by pentacyclic triterpene soap
Glycosides connects what two glucuronic acids were constituted by glycosidic bond.Glycyrrhizic acid removes its end through beta-glucuronidase enzyme hydrolysis
One glucuronic acid base is generated as liquorice enoxolone (such as Fig. 3).
Liquorice enoxolone is again GAMG (GAMG).The using value of liquorice enoxolone (GAMG) is far away
More than glycyrrhizic acid.Liquorice enoxolone sugariness is 5 times of glycyrrhizic acid, is 941 times of sucrose, is a kind of high sugariness, low in calories novel
Sweetener, is effectively reduced the diseases such as the obesity taking in initiation because of high heat sweetener, diabetes, hyperlipemia, carious tooth;Sweet
Grass time glycosides stable in properties, high temperature resistant, acidproof, high pressure resistant, wider in field of food range of application;As medicine, liquorice enoxolone and
Glycyrrhizic acid has the pharmacologically active of identical (or higher), as having anti-inflammatory, antiviral, antitumor, Antiulcer activity, anti-mistake
Quick, protect the liver, the effect such as reducing blood lipid, but liquorice enoxolone middle polarity, solubility preferably and is easier to transmembrane transport in vivo, and ratio is sweet
Oxalic acid bioavilability is more preferable;The most important thing is that liquorice enoxolone is more safer than glycyrrhizic acid, the LD50 of liquorice enoxolone is 5000mg/
Kg, and the LD50 of glycyrrhizic acid is 805mg/kg.Therefore produce and exploitation liquorice enoxolone has critically important using value and reality
Meaning.
Kanaoka M chemical synthesis synthesize liquorice enoxolone (M Kanaoka, Chem pharm Bull (Tokyo),
1986,34 (12): 4978~4983.) but synthetic route is long, yield is low.Traditional chemical hydrolysis removes Radix Glycyrrhizae by hydrolysis
The glucuronic acid base of acid generates liquorice enoxolone, but this method is selectively low to two glycosidic bonds of glycyrrhizic acid, it is impossible to directionally hydrolyzing
Generating liquorice enoxolone, accessory substance is many, and yield is relatively low, there is highly energy-consuming simultaneously, the shortcoming of high pollution.Additionally, according to European Union and U.S.
The legislation of state, the product being obtained by chemical method is not natural materials, is applied to field of food and is restricted, and utilizes enzyme process
Or the material that obtains of microbial method (i.e. biotransformation method) is it is considered to be natural materials.Bioconversion refers to utilize microorganism, moves
Plant and cultivating system or its enzyme producing carry out the biochemical reaction process of structural modification to xenobiontics, and its essence is
The enzyme utilizing living things system to produce carries out enzymic catalytic reaction to xenobiontics.Biotransformation method conversion glycyrrhizic acid generates Radix Glycyrrhizae time
Glycosides also has been reported that.According to it has been reported that enteric bacteria, animal tissue is contained beta-glucuronidase enzyme, Radix Glycyrrhizae can be converted
Acid generates liquorice enoxolone but it generally exists that the selectivity of conversion is on the low side (not merely to be produced liquorice enoxolone, also produce substantial amounts of pair
Product), the relatively low shortcoming of transformation efficiency.Extracting beta-glucuronidase enzyme from animal tissue, cost is high especially, prepares numerous
Trivial.
The present inventor separates from grass Dongxiang Wild Rice leaf tissue can orient conversion glycyrrhizic acid
Generating the endogenetic fungus Aspergillus flavus DX-SEL2 of liquorice enoxolone, this bacterium has been preserved in Chinese Typical Representative and has cultivated
Thing preservation center, preserving number is CCTCCNO:M 2013686.This bacterium can be determined under the induction of glycyrrhizic acid (or ammonium glycyrrhizunate)
Generating liquorice enoxolone to conversion glycyrrhizic acid, and conversion ratio being higher, step of converting is simple, and environmental protection is pollution-free.
It additionally, present invention firstly discovers that endogenetic fungus conversion glycyrrhizic acid generates liquorice enoxolone, and is to have no to close with Radix Glycyrrhizae
The endogenetic fungus of the grass Dongxiang Wild Rice of connection, this provides new research think of for finding related Efficient Conversion bacterial strain
Road.
Content of the invention
It is an object of the invention to provide a strain Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 to turn
Glycyrrhizic acid orientation generates liquorice enoxolone, overcomes catalysis directionality difference of the prior art, pollutes environment, highly energy-consuming, conversion
Rate is low waits deficiency.
The conversion glycyrrhizic acid of the present invention generates the Dongxiang Wild Rice endogenetic fungus of liquorice enoxolone, and its Classification And Nomenclature is
Aspergillus flavus DX-SEL2, is to use endogenetic fungus to separate from the leaf tissue of Dongxiang Wild Rice plant living body
Purification technique separates and obtains, and has been preserved in China typical culture collection center, and preservation date is on December 23rd, 2013, preservation
Number be CCTCC NO:M 2013686.
Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 morphological feature of the present invention is:
On PDA culture medium, cultivating 2 days for 28 ± 2 DEG C, colony diameter 30~32mm, within 3 days, colony diameter is 61~63mm, within 4 days, covers with whole
Culture dish;Quality velvet shape, thicker, central authorities are existing cotton-shaped, and bacterium colony front is olive-green, and edge is slightly white;Reverse side is faint yellow.Point
Raw sporophore is colourless or light brown, a diameter of 10~15 m;Conidium top capsule is almost spherical, and a diameter of 50~65 m are mitogenetic
Spore is spherical to subsphaeroidal, a diameter of 2.5~4.5 m.
Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 gene accession number of the present invention is
KC871017。
Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 of the present invention converts Radix Glycyrrhizae acid cut
Comprise the following steps to generating liquorice enoxolone:
Step one, by Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2, be inoculated into PDA inclined-plane training
Support and base is cultivated 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 cultivated to exponential phase.
Step 2, the inoculum concentration according to 10%~30% are seeded to the seed liquor of above-mentioned exponential phase containing glycyrrhizic acid
In conversion culture medium, conversion produces liquorice enoxolone to content substantially constant.
Step 3, from zymotic fluid isolated and purified liquorice enoxolone.
Preferably, described Aspergillus flavus DX-SEL2 conversion glycyrrhizic acid orientation generates liquorice enoxolone,
The consisting of of seed culture medium in step one: 10~40g containing glucose in every liter of culture medium, glycyrrhizic acid (or ammonium glycyrrhizunate)
0.1~1g, KH2PO41.0~3.0g, NH4NO32.0~5.0g, NaCl 0.3~0.8 g, dusty yeast 0.05~0.3 g,
MgSO40.1~0.5g, CaCl20.01~0.5g, FeSO40.014g, ZnSO4·7H2O 2.9mg, MnCl2·4H2O
2.0mg, CuSO4·5H2O 0.25mg, CoCl2·6H2O 0.24mg, Na2MoO4·2H2O 0.24mg, H3BO30.03mg,
Remaining is pure water, adjusts pH to be 5.0~7.0.
Preferably, described Aspergillus flavus DX-SEL2 conversion glycyrrhizic acid orientation generates liquorice enoxolone,
Convert culture medium to consist of: every liter of culture medium contains glycyrrhizic acid (or ammonium glycyrrhizunate) 2~30g, KH in step 22PO4
1.0~3.0g, NH4NO32.0~4.0g, NaCl 0.3~0.8 g, dusty yeast 0.05~0.1g, MgSO40.1~
0.5g, CaCl20.01~0.5g, FeSO40.014g, ZnSO4·7H2O 2.9mg, MnCl2·4H2O 2.0mg,
CuSO4·5H2O 0.25mg, CoCl2·6H2O 0.24mg, Na2MoO4·2H2O 0.24mg, H3BO30.03mg, remaining
For pure water, pH is adjusted to be 3.5~9.0.Wherein glycyrrhizic acid (or ammonium glycyrrhizunate) is the induction that thalline produces beta-glucuronidase enzyme
Agent.
Preferably, described Aspergillus flavus DX-SEL2 conversion glycyrrhizic acid orientation generates liquorice enoxolone,
In step 2, condition of culture is: 28 DEG C~60 DEG C, rotating speed 150~300r/min,.
Preferably, described Aspergillus flavus DX-SEL2 conversion glycyrrhizic acid orientation generates liquorice enoxolone,
In step 3, the isolated and purified of liquorice enoxolone comprises the steps of
Buchner funnel suction filtration or six layers of filtered through gauze, collect filtrate, 2~4 times with the extraction of chloroform equal-volume (extracts and polarity relatively
Low impurity, liquorice enoxolone without extracting).Aqueous phase continues to extract 3~5 times with ethyl acetate equal-volume, obtains ethyl acetate
Phase reduced pressure concentration is liquorice enoxolone crude product.Then it is further purified by methods such as purification on normal-phase silica gel column chromatography or reversed phase column chromatographies again
Liquorice enoxolone.
Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 of the present invention converts Radix Glycyrrhizae acid cut
To having the advantages that of generation liquorice enoxolone: the enzyme producing with microorganism makees catalyst, reaction efficiency is high, selectivity
By force, accessory substance is few, and step is simple, and environmental protection is pollution-free.
Brief description
Fig. 1 is the bacterium colony shape of Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 of the present invention
State;
Fig. 2 is under Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 ESEM of the present invention
Sporangium and spore shape
Fig. 3 glycyrrhizic acid is generated liquorice enoxolone by beta-glucuronidase enzyme hydrolysis
Fig. 4 is Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 of the present invention conversion glycyrrhizic acid
The HPLC detection of product;
The LC-MS detection of Fig. 5 endogenetic fungus Aspergillus flavus DX-SEL2 conversion glycyrrhizic acid product.
Detailed description of the invention
Carry out describing in detail bright to the present invention below in conjunction with example.
Embodiment 1: the separation of Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 of the present invention
(1) gather complete Dongxiang Wild Rice, return to laboratory and immediately treat.
(2) root, stem and leaf all use 75% ethanol leaching 5min surface primary sterilization, then soak with 0.1% mercuric chloride
8min, the mode of rinsed with sterile water tissue surface sterilizing.
(3) two is cut by the above-mentioned stem handled well the scissors sterilizing, then the stem vertical profile at two will be removed, one point
It is two, then be cut into the fritter of 0.1 × 0.5cm.The edge of leaf is cut off by sterilized scissors, the blade having cut off edge
Also it is cut into the fritter of about 0.2 × 0.5cm.They are respectively placed in the PDA of additional 60 μ g/L streptomysins and 0.5g/L potassium bichromate
On culture medium, put in 28 DEG C of incubators and cultivate.In order to check surface sterilization effect, if comparison carries out the inspection of sterilization effect.Right
According to I: the root carrying out disinfecting action, stem and leaf are not cut off two and edge, then by their surface and solid plate
Take out after contact, culture dish is put into incubator and cultivates;Comparison II: last washing sterilized water accesses in culture medium.Often
Individual process is repeated 3 times.
(4) discovery inoculation tissue around grows bacterium colony, with transfer needle picking mycelia front end, is inoculated into another culture medium
On.Every day observes once, if growing bacterium colony, chooses immediately.
(5), after the fungi chosen forms bacterium colony, with transfer needle picking mycelia front end, inoculate on another culture medium,
So repeatedly purify 4 times, bacterial classification is received 4 DEG C of preservations on slant medium
Embodiment 2: the present invention filters out the Dongxiang Wild Rice endogenetic fungus that orientation conversion glycyrrhizic acid generates liquorice enoxolone
Aspergillus flavus DX-SEL2。
(1) flat board primary dcreening operation: by the endogenetic fungus activation of the above-mentioned Dongxiang Wild Rice being separated to, be inoculated in glycyrrhizic acid conduct
In the solid screening and culturing base of sole carbon source, control group glucose replaces glycyrrhizic acid as sole carbon source.28 DEG C, cultivate 7d,
Filter out and can give birth to well-grown bacterial strain.
Described screening and culturing base is;G, KH containing glycyrrhizic acid 3.0 in every liter of culture medium2PO42.2 g, NH4NO3 3.0
G, NaCl 0.5 g, dusty yeast 0.05 g, MgSO40.12g, CaCl20.014g, FeSO40.014g, ZnSO4·7H2O
2.9mg, MnCl2·4H2O 2.0mg, CuSO4·5H2O 0.25mg, CoCl2·6H2O 0.24mg, Na2MoO4·2H2O
0.24mg, H3BO30.03mg, agar powder 20g(fluid nutrient medium is not added with), remaining is pure water, pH6.0.
(2) shaking flask is sieved again: by above-mentioned flat board primary dcreening operation inoculation out to seed culture medium (with the glucose of 20g/L
Replace the glycyrrhizic acid of the 3.0g/L in screening and culturing base) in 28 DEG C, 150 rpm cultivate 2d, then by 20% inoculum concentration switching
It is in the liquid screening medium of sole carbon source to glycyrrhizic acid, and sets two comparisons: a liquid screening medium being does not connects
Enter thalline, to detect whether glycyrrhizic acid decomposes in the medium), two be bacterial strain is transferred to glucose replace glycyrrhizic acid as
In the screening and culturing base of sole carbon source.28 DEG C, 150 rpm shaking tables cultivation 4d, collect zymotic fluid, with TLC, HPLC and LC~Ms
The product of detection conversion.
Test result indicate that: Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 can orient and be converted into
Liquorice enoxolone (such as Fig. 4, Fig. 5), ((GL molar concentration after initial GL molar concentration~conversion)/initial GL's glycyrrhizic acid conversion ratio rubs
That concentration × 100%) it is 72.4%, liquorice enoxolone productivity (GAMG molar concentration/initial GL molar concentration × 100% after conversion) is
67.3%。
Embodiment 3: endogenetic fungus Aspergillus flavus DX-SEL2 conversion glycyrrhizic acid generates liquorice enoxolone
Dongxiang Wild Rice endogenetic fungus Aspergillus flavus DX-SEL2 after activation is linked into seed culture
28 DEG C of base, 250 rpm cultivate 3d, are then forwarded in conversion culture medium by the inoculum concentration of 30%, 35 DEG C, 250 rpm shaking table trainings
Support 7d.The centrifugal thalline that goes, collection zymotic fluid, detects converted product with HPLc.
Consisting of of described seed culture medium: 25g containing glucose in every liter of culture medium, glycyrrhizic acid (or ammonium glycyrrhizunate)
0.2g, KH2PO42.0g, NH4NO33g, NaCl 0.5g, dusty yeast 0.1g, MgSO40.12g, CaCl20.2g, FeSO4
0.014g, ZnSO4·7H2O 2.9mg, MnCl2·4H2O 2.0mg, CuSO4·5H2O 0.25mg, CoCl2·6H2O
0.24mg, Na2MoO4·2H2O 0.24mg, H3BO30.03mg, remaining is pure water, adjusts pH to be 6.0.
Described conversion culture medium consists of: 5g containing mono-ammonium glycyrrhizinate, KH in every liter of culture medium2PO42.2g, NH4NO3
3g, NaCl 0.5g, dusty yeast 0.1 g, MgSO40.12g, CaCl20.4g, FeSO40.014g, ZnSO4·7H2O
2.9mg, MnCl2·4H2O 2.0mg, CuSO4·5H2O 0.25mg, CoCl2·6H2O 0.24mg, Na2MoO4·2H2O
0.24mg, H3BO30.03mg, remaining is pure water, adjusts pH to be 7.0.
After conversion terminates, suction filtration goes thalline obtain filtrate and detect converted product, and testing result is that glycyrrhizic acid conversion ratio is
90.5%, liquorice enoxolone productivity is 85.3%.
Gained filtrate extracts 3 relatively low impurity of extraction depolarization with chloroform equal-volume, liquorice enoxolone without extracting.Aqueous phase
Continue to extract 3~5 times with ethyl acetate equal-volume, obtain ethyl acetate phase reduced pressure concentration and be liquorice enoxolone crude product, detect pure
Degree is 84.8%.
Claims (4)
1. a strain conversion glycyrrhizic acid generates the Dongxiang Wild Rice endogenetic fungus of liquorice enoxolone, it is characterized in that: described endogenetic fungus
It is to use endogenetic fungus separating and purifying technology to separate from the leaf tissue of Dongxiang Wild Rice plant living body to obtain, divide through microorganism
Class identifies named Aspergillus flavus DX-SEL2, has been preserved in China typical culture collection center, preservation
Number be CCTCC NO:M 2013686.
2. endogenetic fungus conversion glycyrrhizic acid as claimed in claim 1 generates the method for liquorice enoxolone, comprises the following steps:
Step one, described endogenetic fungus is inoculated in PDA slant medium cultivation activation 72~84 hours, and makes spore
Suspension, is then seeded in seed culture medium, 25~35 DEG C, 150~300r/min, and shaking table is cultivated to exponential phase;Its
Consisting of of middle seed culture medium: 10~40g containing glucose in every liter of culture medium, glycyrrhizic acid or ammonium glycyrrhizunate 0.1~1g,
KH2PO41.0~3.0g, NH4NO32.0~5.0g, NaCl 0.3~0.8g, dusty yeast 0.05~0.3g, MgSO40.1~
0.5g, CaCl20.01~0.5g, FeSO40.014g, ZnSO4·7H2O 2.9mg, MnCl2·4H2O 2.0mg, CuSO4·5H2O
0.25mg, CoCl2·6H2O 0.24mg, Na2MoO4·2H2O 0.24mg, H3BO30.03mg, remaining is pure water, and tune pH is
5.0~7.0;
Step 2, the inoculum concentration according to 10%~40% are seeded to the conversion containing glycyrrhizic acid the seed liquor of above-mentioned exponential phase
In culture medium, conversion produces liquorice enoxolone to content substantially constant;Wherein convert culture medium to consist of: every liter of culture medium contains sweet
Oxalic acid or ammonium glycyrrhizunate 2~30g, KH2PO41.0~3.0g, NH4NO32.0~4.0g, NaCl 0.3~0.8g, dusty yeast
0.05~0.1g, MgSO40.1~0.5g, CaCl20.01~0.5g, FeSO40.014g, ZnSO4·7H2O 2.9mg,
MnCl2·4H2O 2.0mg, CuSO4·5H2O 0.25mg, CoCl2·6H2O 0.24mg, Na2MoO4·2H2O 0.24mg,
H3BO30.03mg, remaining is pure water, adjusts pH to be 3.5~9.0;Wherein glycyrrhizic acid or ammonium glycyrrhizunate are that thalline produces β-glucose
The derivant of aldehyde neuraminidase;
Step 3, from zymotic fluid isolated and purified liquorice enoxolone.
3. method as claimed in claim 2, is characterized in that, in step 2, condition of culture is: 28 DEG C~35 DEG C, rotating speed 150
~300r/min.
4. method as claimed in claim 2, is characterized in that, in step 3, the isolated and purified of liquorice enoxolone comprises following step
Rapid:
Buchner funnel suction filtration or six layers of filtered through gauze, collect filtrate, extracts 2~4 times with chloroform equal-volume, and aqueous phase continues to use acetic acid
Ethyl ester equal-volume extracts 3~5 times, obtains ethyl acetate phase reduced pressure concentration and is liquorice enoxolone crude product, then uses purification on normal-phase silica gel again
Column chromatography or reversed phase column chromatography method are further purified liquorice enoxolone.
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CN106701604B (en) * | 2017-03-24 | 2020-09-15 | 江西科技师范大学 | Dongxiang wild rice endophytic fungus for efficiently converting glycyrrhizic acid to produce GAMG and application thereof |
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