CN103992953A - Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide - Google Patents
Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide Download PDFInfo
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- CN103992953A CN103992953A CN201410057606.9A CN201410057606A CN103992953A CN 103992953 A CN103992953 A CN 103992953A CN 201410057606 A CN201410057606 A CN 201410057606A CN 103992953 A CN103992953 A CN 103992953A
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- starrhizin
- potenlini
- endogenetic fungus
- transforms
- sel2
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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 62
- 241000233866 Fungi Species 0.000 title claims abstract description 29
- 230000001131 transforming effect Effects 0.000 title abstract description 3
- 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 title abstract 8
- 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 title abstract 4
- 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 abstract 4
- 229960003720 enoxolone Drugs 0.000 title abstract 4
- 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 abstract 4
- 229960004949 glycyrrhizic acid Drugs 0.000 title abstract 4
- 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 abstract 4
- 239000001685 glycyrrhizic acid Substances 0.000 title abstract 4
- 235000019410 glycyrrhizin Nutrition 0.000 title abstract 4
- 241000746966 Zizania Species 0.000 title abstract 2
- 235000002636 Zizania aquatica Nutrition 0.000 title abstract 2
- 238000006243 chemical reaction Methods 0.000 claims abstract description 13
- 102000053187 Glucuronidase Human genes 0.000 claims abstract description 7
- 108010060309 Glucuronidase Proteins 0.000 claims abstract description 7
- 238000000746 purification Methods 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 6
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 4
- 230000000813 microbial effect Effects 0.000 claims abstract description 3
- HLDYLAJAWSKPFZ-QDPIGISRSA-N glycyrrhetic acid 3-O-glucuronide Chemical compound O([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@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O HLDYLAJAWSKPFZ-QDPIGISRSA-N 0.000 claims description 55
- 241000228212 Aspergillus Species 0.000 claims description 26
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 238000011218 seed culture Methods 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 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
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000008103 glucose 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
- 238000005516 engineering process Methods 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000002054 inoculum Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 2
- 238000004440 column chromatography Methods 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 238000012797 qualification Methods 0.000 claims 1
- 230000009466 transformation Effects 0.000 abstract description 7
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 5
- 230000007062 hydrolysis Effects 0.000 abstract description 4
- 230000007613 environmental effect Effects 0.000 abstract description 3
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- 241000228197 Aspergillus flavus Species 0.000 abstract 1
- 230000003197 catalytic effect Effects 0.000 abstract 1
- 238000000855 fermentation Methods 0.000 abstract 1
- 230000004151 fermentation Effects 0.000 abstract 1
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- 239000003960 organic solvent Substances 0.000 abstract 1
- 238000000638 solvent extraction Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000000284 extract Substances 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
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- 239000006227 byproduct Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- YVECGMZCTULTIS-PBXRRBTRSA-N glucal Chemical compound OC[C@H]1OC=C[C@@H](O)[C@@H]1O YVECGMZCTULTIS-PBXRRBTRSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
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- 229920001817 Agar Polymers 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 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
- 208000008589 Obesity Diseases 0.000 description 1
- -1 Streptomycin sulphates Chemical class 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- 230000028161 membrane depolarization Effects 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- VIKNJXKGJWUCNN-XGXHKTLJSA-N norethisterone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@](CC4)(O)C#C)[C@@H]4[C@@H]3CCC2=C1 VIKNJXKGJWUCNN-XGXHKTLJSA-N 0.000 description 1
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- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a Dongxiang wild rice endophytic fungus strain capable of transforming glycyrrhizic acid to produce glycyrrhetinic acid monoglucuronide. The strain is identified and named as Aspergillus flavus DX-SEL2 and preserved in China Center for Type Culture Collection, with an accession number of CCTCC NO: M2013686. The strain can induce generation of beta-glucuronidase in a transformation medium with a glycyrrhizic acid as an inductive agent, then hydrolysis is carried out so as to remove a glucuronyl group at the terminal of a glycyrrhizic acid molecule, so glycyrrhetinic acid monoglucuronide is produced (secreted in fermentation broth). Organic solvent extraction, normal-phase silica gel column chromatography, reversed-phase silica gel column chromatography and the like are used for further separation and purification so as to obtain glycyrrhetinic acid monoglucuronide. According to the invention, microbial catalytic conversion is employed, and the advantages of greenness, environmental protection, no pollution, simple transformation steps, a single product, a high transformation rate, etc. are obtained.
Description
Technical field
The present invention relates to conversion technology field, particularly relates to the Dongxiang Wild Rice endogenetic fungus of a strain conversion Potenlini generation Starrhizin.
Background technology
Potenlini (GL) is one of main active ingredient in Radix Glycyrrhizae.The structure of Potenlini, as Fig. 3, connects two glucuronic acids by pentacyclic triterpene saponin by glycosidic link and forms.The glucal acidic group that Potenlini is removed its end through beta-glucuronidase enzymic hydrolysis just generates Starrhizin (as Fig. 3).
Starrhizin is again GAMG (GAMG).The using value of Starrhizin (GAMG) is far longer than Potenlini.Starrhizin sugariness is 5 times of Potenlini, is 941 times of sucrose, is a kind of high sugariness, novel sweetener low in calories, can effectively reduce because of diseases such as obesity that the absorption of high calorie sweeting agent causes, diabetes, hyperlipidemia, carious teeth; Starrhizin stable in properties, high temperature resistant, acidproof, high pressure resistant, wider in field of food range of application; As medicine, Starrhizin and Potenlini have the pharmacologically active of identical (or stronger), as there is anti-inflammatory, antiviral, antitumor, Antiulcer activity, antianaphylaxis, protect the liver, the effect such as reducing blood-fat, but Starrhizin middle polarity, solubleness is preferably and easier transmembrane transport, better than Potenlini bioavailability in vivo; The most important thing is that Starrhizin is more safer than Potenlini, the LD50 of Starrhizin is 5000mg/kg, and the LD50 of Potenlini is 805mg/kg.Therefore produce and develop Starrhizin and there is very important using value and realistic meaning.
4978～4983.) but synthetic route is long the synthetic Starrhizin of chemical synthesis for Kanaoka M (M Kanaoka, Chem pharm Bull (Tokyo), 1986,34 (12):, yield is low.The glucal acidic group that traditional chemical hydrolysis removes Potenlini by hydrolysis generates Starrhizin, but this method is low to two of Potenlini glycosidic link selectivity, can not generate Starrhizin by directionally hydrolyzing, by product is many, yield is lower, has highly energy-consuming simultaneously, the shortcoming of high pollution.In addition,, according to the legislation of European Union and the U.S., the product obtaining by chemical process is not crude substance, be applied to field of food and be restricted, and the material that utilizes enzyme process or microbial method (being biotransformation method) to obtain is considered to crude substance.Bio-transformation refers to and utilizes the enzyme of microorganism, animals and plants and culture system or its generation xenobiontics to be carried out to the biochemical reaction process of structural modification, and its essence is that the enzyme that utilizes living things system to produce carries out enzymic catalytic reaction to xenobiontics.Biotransformation method transforms Potenlini generation Starrhizin also report.According to having been reported, in intestinal bacteria, animal tissues, contain beta-glucuronidase enzyme, Potenlini be can transform and the specificity (not merely produce Starrhizin, also produce a large amount of by products) on the low side that Starrhizin but its ubiquity transform, the shortcoming that transformation efficiency is lower generated.From animal tissues, extract beta-glucuronidase enzyme, cost is high especially, prepares loaded down with trivial details.
The inventor separates the endogenetic fungus that can directedly transform Potenlini generation Starrhizin from grass Dongxiang Wild Rice leaf tissue---
aspergillus flavusdX-SEL2, this bacterium has been preserved in Chinese Typical Representative culture collection center, and preserving number is CCTCCNO:M 2013686.This bacterium can generate Starrhizin by the directed Potenlini that transforms under the induction of Potenlini (or ammonium glycyrrhizunate), and transformation efficiency is higher, and step of converting is simple, and environmental protection is pollution-free.
In addition, the present invention finds that endogenetic fungus transforms Potenlini and generates Starrhizin first, and is and the endogenetic fungus of the uncorrelated grass Dongxiang Wild Rice of Radix Glycyrrhizae milli, and this provides new Research Thinking for finding relevant Efficient Conversion bacterial strain.
Summary of the invention
The object of this invention is to provide a strain Dongxiang Wild Rice endogenetic fungus
aspergillus flavusdX-SEL2 transforms the directed Starrhizin that generates of Potenlini, has overcome catalysis directional property of the prior art poor, contaminate environment, highly energy-consuming, the deficiencies such as low conversion rate.
Conversion Potenlini of the present invention generates the Dongxiang Wild Rice endogenetic fungus of Starrhizin, and its Classification And Nomenclature is
aspergillus flavusdX-SEL2, from the leaf tissue of Dongxiang Wild Rice plant living body, to adopt endogenetic fungus separating and purifying technology to separate to obtain, be preserved in Chinese Typical Representative culture collection center, preservation date is on December 23rd, 2013, and preserving number is CCTCC NO:M 2013686.
Dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusdX-SEL2 morphological specificity is: on PDA substratum, cultivates 2 days for 28 ± 2 DEG C, and colony diameter 30～32mm, 3 days colony diameters are 61～63mm, within 4 days, cover with whole culture dish; Quality velvet shape, thicker, central authorities are existing cotton-shaped, and bacterium colony front is olive-green, and edge is white slightly; Reverse side is faint yellow.Colourless or the light brown of conidiophore, diameter is 10～15 μ m; Conidium top capsule is close to spherical, and diameter is 50～65 μ m, and conidium is spherical to subsphaeroidal, and diameter is 2.5～4.5 μ m.
Dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusdX-SEL2 gene accession number is KC871017.
Dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusdX-SEL2 transforms the directed Starrhizin that generates of Potenlini and comprises the following steps:
Step 1, by Dongxiang Wild Rice endogenetic fungus
aspergillus flavusdX-SEL2, is inoculated in PDA slant medium and cultivates and activate 72～84 hours, and make spore suspension, is then seeded in seed culture medium, and 28 ± 1 DEG C, 150～300r/min, shaking table is cultured to logarithmic phase.
Step 2, inoculum size according to 10%～30% are seeded to the seed liquor of above-mentioned logarithmic phase in the conversion substratum containing Potenlini, transform and produce Starrhizin to content substantially constant.
Step 3, from fermented liquid separation and purification Starrhizin.
Preferably, described
aspergillus flavusdX-SEL2 transforms the directed Starrhizin that generates of Potenlini, the consisting of of seed culture medium in step 1: in every liter of substratum containing glucose 10～40g, Potenlini (or ammonium glycyrrhizunate) 0.1～1g, KH
2pO
41.0～3.0g, NH
4nO
32.0～5.0g, NaCl 0.3～0.8 g, yeast powder 0.05～0.3 g, MgSO
40.1～0.5g, CaCl
20.01～0.5g, FeSO
40.014g, ZnSO
47H
2o 2.9mg, MnCl
24H
2o 2.0mg, CuSO
45H
2o 0.25mg, CoCl
26H
2o 0.24mg, Na
2moO
42H
2o 0.24mg, H
3bO
30.03mg, all the other are pure water, adjusting pH is 5.0～7.0.
Preferably, described
aspergillus flavusdX-SEL2 transforms the directed Starrhizin that generates of Potenlini, transforms substratum and consists of: in every liter of substratum, contain Potenlini (or ammonium glycyrrhizunate) 2～30g, KH in step 2
2pO
41.0～3.0g, NH
4nO
32.0～4.0g, NaCl 0.3～0.8 g, yeast powder 0.05～0.1g, MgSO
40.1～0.5g, CaCl
20.01～0.5g, FeSO
40.014g, ZnSO
47H
2o 2.9mg, MnCl
24H
2o 2.0mg, CuSO
45H
2o 0.25mg, CoCl
26H
2o 0.24mg, Na
2moO
42H
2o 0.24mg, H
3bO
30.03mg, all the other are pure water, adjusting pH is 3.5～9.0.Wherein Potenlini (or ammonium glycyrrhizunate) is the inductor that thalline produces beta-glucuronidase enzyme.
Preferably, described
aspergillus flavusdX-SEL2 transforms the directed Starrhizin that generates of Potenlini, and in step 2, culture condition is: 28 DEG C～60 DEG C, and rotating speed 150～300r/min.
Preferably, described
aspergillus flavusdX-SEL2 transforms the directed Starrhizin that generates of Potenlini, and in step 3, the separation and purification of Starrhizin comprises following steps:
Büchner funnel suction filtration or six layers of filtered through gauze, collect filtrate, with 2～4 times (extract and the impurity that polarity is lower, and can not extract and Starrhizin) of chloroform equal-volume extraction.Water continues, with ethyl acetate equal-volume extraction 3～5 times, to obtain ethyl acetate phase concentrating under reduced pressure and be Starrhizin crude product.And then be further purified Starrhizin by methods such as purification on normal-phase silica gel column chromatography or reversed phase column chromatographies.
Dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusdX-SEL2 transforms the directed beneficial effect that generates Starrhizin of Potenlini and is mainly reflected in: make catalyzer with the enzyme of microorganisms, reaction efficiency is high, and specificity is strong, and by product is few, and step is simple, and environmental protection is pollution-free.
Brief description of the drawings
Fig. 1 is Dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusthe colonial morphology of DX-SEL2;
Fig. 2 is Dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusdX-SEL2 scanning electron microscope hyposporangium and spore shape
Fig. 3 Potenlini is generated Starrhizin by beta-glucuronidase enzymic hydrolysis
Fig. 4 is Dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusthe HPLC that DX-SEL2 transforms Potenlini product detects;
Fig. 5 endogenetic fungus
aspergillus flavusthe LC-MS that DX-SEL2 transforms Potenlini product detects.
Embodiment
the present invention is described in detail bright below in conjunction with example.
embodiment 1:dongxiang Wild Rice endogenetic fungus of the present invention
aspergillus flavusthe separation of DX-SEL2
(1) gather complete Dongxiang Wild Rice, get back to laboratory and process immediately.
(2) root, stem and leaf all adopt 75% ethanol to soak the preliminary sterilizing in 5min surface, then by 0.1% mercuric chloride immersion 8min, the mode of rinsed with sterile water tissue surface sterilizing.
(3) the above-mentioned stem of handling well is cut two with the scissors of sterilizing, then by removing the stem vertical profile at two, be divided into two, then be cut into the fritter of 0.1 × 0.5cm.The edge of leaf is cut off with sterilized scissors, the blade that has cut off edge is also cut into the approximately fritter of 0.2 × 0.5cm.They are placed in respectively on the PDA substratum of additional 60 μ g/L Streptomycin sulphates and 0.5g/L potassium bichromate, put in 28 DEG C of incubators and cultivate.In order to check surperficial sterilising effect, establish contrast and carry out the inspection of sterilising effect.Contrast I: the root, stem and the leaf that have carried out disinfecting action are not cut off to two and edge, take out after then their surface being contacted with solid plate, culture dish is put into incubator and cultivate; Contrast II: last washing is with in sterilized water access substratum.Each processing repeats 3 times.
(4) find that inoculation tissue grows bacterium colony around, with inoculating needle picking mycelia front end, be inoculated on another substratum.Observe once every day, if grow bacterium colony, chooses immediately.
(5) fungi of choosing forms after bacterium colony, with inoculating needle picking mycelia front end, inoculates on another substratum, and purifying 4 times so repeatedly, receives 4 DEG C of preservations on slant medium by bacterial classification
embodiment 2:the present invention filters out the directed Dongxiang Wild Rice endogenetic fungus that transforms Potenlini generation Starrhizin
aspergillus flavusdX-SEL2.
(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 the solid screening culture medium of Potenlini as sole carbon source, control group replaces Potenlini as sole carbon source with glucose.28 DEG C, cultivate 7d, filter out and can give birth to well-grown bacterial strain.
Described screening culture medium is; In every liter of substratum, contain Potenlini 3.0 g, KH
2pO
42.2 g, NH
4nO
33.0 g, NaCl 0.5 g, yeast powder 0.05 g, MgSO
40.12g, CaCl
20.014g, FeSO
40.014g, ZnSO
47H
2o 2.9mg, MnCl
24H
2o 2.0mg, CuSO
45H
2o 0.25mg, CoCl
26H
2o 0.24mg, Na
2moO
42H
2o 0.24mg, H
3bO
30.03mg, agar powder 20g(liquid 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 is out arrived to 28 DEG C, 150 rpm cultivation 2d in seed culture medium (replacing the Potenlini of the 3.0g/L in screening culture medium with the glucose of 20g/L), then be forwarded in the liquid screening substratum that Potenlini is sole carbon source by 20% inoculum size, and establish two contrasts: the one, liquid screening substratum in do not access thalline, whether in substratum, decompose to detect Potenlini), the 2nd, bacterial strain is transferred to glucose replacement Potenlini in the screening culture medium of sole carbon source.28 DEG C, 150 rpm shaking tables cultivation 4d, collect fermented liquid, detects the product of conversion with TLC, HPLC and LC～Ms.
Experimental result shows: Dongxiang Wild Rice endogenetic fungus
aspergillus flavusdX-SEL2 can orientation be converted into Starrhizin (as Fig. 4, Fig. 5), Potenlini transformation efficiency ((GL volumetric molar concentration after initial GL volumetric molar concentration～conversion)/initial GL volumetric molar concentration × 100%) is 72.4%, and Starrhizin productive rate (GAMG volumetric molar concentration/initial GL volumetric molar concentration × 100% after transforming) is 67.3%.
embodiment 3:endogenetic fungus
aspergillus flavusdX-SEL2 transforms Potenlini and generates Starrhizin
By the Dongxiang Wild Rice endogenetic fungus after activation
aspergillus flavusdX-SEL2 is linked into 28 DEG C of seed culture mediums, 250 rpm cultivate 3d, is then forwarded to and transforms in substratum by 30% inoculum size, 35 DEG C, 250 rpm shaking tables cultivation 7d.The centrifugal thalline that goes, collects fermented liquid, detects converted product with HPLc.
Consisting of of described seed culture medium: contain glucose 25g, Potenlini (or ammonium glycyrrhizunate) 0.2g, KH in every liter of substratum
2pO
42.0g, NH
4nO
33g, NaCl 0.5g, yeast powder 0.1g, MgSO
40.12g, CaCl
20.2g, FeSO
40.014g, ZnSO
47H
2o 2.9mg, MnCl
24H
2o 2.0mg, CuSO
45H
2o 0.25mg, CoCl
26H
2o 0.24mg, Na
2moO
42H
2o 0.24mg, H
3bO
30.03mg, all the other are pure water, adjusting pH is 6.0.
Described conversion substratum consists of: in every liter of substratum, contain monoammonium glycyrrhizinate 5g, KH
2pO
42.2g, NH
4nO
33g, NaCl 0.5g, yeast powder 0.1 g, MgSO
40.12g, CaCl
20.4g, FeSO
40.014g, ZnSO
47H
2o 2.9mg, MnCl
24H
2o 2.0mg, CuSO
45H
2o 0.25mg, CoCl
26H
2o 0.24mg, Na
2moO
42H
2o 0.24mg, H
3bO
30.03mg, all the other are pure water, adjusting pH is 7.0.
After conversion finishes, suction filtration goes thalline to obtain filtrate and detects converted product, and detected result is that Potenlini transformation efficiency is 90.5%, and Starrhizin productive rate is 85.3%.
Gained filtrate extracts the lower impurity of depolarization 3 times with the extraction of chloroform equal-volume, and can not extract and Starrhizin.Water continues, with ethyl acetate equal-volume extraction 3～5 times, to obtain ethyl acetate phase concentrating under reduced pressure and be Starrhizin crude product, and detecting purity is 84.8%.
Claims (8)
1.
one strain transforms the Dongxiang Wild Rice endogenetic fungus of Potenlini generation Starrhizin, it is characterized in that: described endogenetic fungus is from the leaf tissue of Dongxiang Wild Rice plant living body, to adopt endogenetic fungus separating and purifying technology to separate to obtain, through microbial taxonomy qualification called after
aspergillus flavusdX-SEL2, has been preserved in Chinese Typical Representative culture collection center, and preservation date is on December 23rd, 2013, and preserving number is CCTCCNO:M 2013686, and this bacterium can transform Potenlini and generate Starrhizin.
2. as claimed in claim 1
aspergillus flavusdX-SEL2 bacterial strain, is characterized in that: on PDA substratum, cultivates 2 days for 28 ± 1 DEG C, and colony diameter 30～32mm, 3 days colony diameters are 61～63mm, within 4 days, cover with whole culture dish; Quality velvet shape, thicker, central authorities are existing cotton-shaped, and bacterium colony front is olive-green, and edge is white slightly; Reverse side is faint yellow, the colourless or light brown of conidiophore, and diameter is 10～15 μ m; Conidium top capsule is close to spherical, and diameter is 50～65 μ m, and conidium is spherical to subsphaeroidal, and diameter is 2.5～4.5 μ m.
3. as claimed in claim 1
aspergillus flavusdX-SEL2 bacterial strain, is characterized in that: its gene accession number is KC871017.
4. endogenetic fungus as claimed in claim 1
aspergillus flavusdX-SEL2 transforms the method for Potenlini generation Starrhizin, it is characterized in that comprising the following steps:
Step 1, by endogenetic fungus
aspergillus flavusdX-SEL2, is inoculated in PDA slant medium and cultivates and activate 72～84 hours, and make spore suspension, is then seeded in seed culture medium, and 25～35 DEG C, 150～300r/min, shaking table is cultured to logarithmic phase;
Step 2, inoculum size according to 10%～40% are seeded to the seed liquor of above-mentioned logarithmic phase in the conversion substratum containing Potenlini, transform and produce Starrhizin to content substantially constant;
Step 3, from fermented liquid separation and purification Starrhizin.
5. endogenetic fungus as claimed in claim 4 transforms Potenlini and generates the method for Starrhizin, it is characterized in that the consisting of of seed culture medium in step 1: in every liter of substratum containing glucose 10～40g, Potenlini (or ammonium glycyrrhizunate) 0.1～1g, KH
2pO
41.0～3.0g, NH
4nO
32.0～5.0g, NaCl 0.3～0.8 g, yeast powder 0.05～0.3 g, MgSO
40.1～0.5g, CaCl
20.01～0.5g, FeSO
40.014g, ZnSO
47H
2o 2.9mg, MnCl
24H
2o 2.0mg, CuSO
45H
2o 0.25mg, CoCl
26H
2o 0.24mg, Na
2moO
42H
2o 0.24mg, H
3bO
30.03mg, all the other are pure water, adjusting pH is 5.0～7.0.
6. endogenetic fungus as claimed in claim 4 transforms the method for Potenlini generation Starrhizin, it is characterized in that, transforms substratum and consist of in step 2: in every liter of substratum, contain Potenlini (or ammonium glycyrrhizunate) 2～30g, KH
2pO
41.0～3.0g, NH
4nO
32.0～4.0g, NaCl 0.3～0.8 g, yeast powder 0.05～0.1g, MgSO
40.1～0.5g, CaCl
20.01～0.5g, FeSO
40.014g, ZnSO
47H
2o 2.9mg, MnCl
24H
2o 2.0mg, CuSO
45H
2o 0.25mg, CoCl
26H
2o 0.24mg, Na
2moO
42H
2o 0.24mg, H
3bO
30.03mg, all the other are pure water, adjusting pH is 3.5～9.0; Wherein Potenlini (or ammonium glycyrrhizunate) is the inductor that thalline produces beta-glucuronidase enzyme.
7. endogenetic fungus as claimed in claim 4 transforms the method for Potenlini generation Starrhizin, it is characterized in that, in step 2, culture condition is: 28 DEG C～60 DEG C, and rotating speed 150～300r/min.
8. endogenetic fungus as claimed in claim 4 transforms the method for Potenlini generation Starrhizin, it is characterized in that, in step 3, the separation and purification of Starrhizin comprises following steps: Büchner funnel suction filtration or six layers of filtered through gauze, collect filtrate, with chloroform equal-volume extraction 2～4 times, water continues with ethyl acetate equal-volume extraction 3～5 times, obtain ethyl acetate phase concentrating under reduced pressure and be Starrhizin crude product, and then be further purified Starrhizin by methods such as purification on normal-phase silica gel column chromatography or reversed phase column chromatographies.
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| CN106047839A (en) * | 2016-04-19 | 2016-10-26 | 北京理工大学 | Fermentation medium and method for improving activity of beta-glucuronidase produced by fungi |
| CN106701604A (en) * | 2017-03-24 | 2017-05-24 | 江西科技师范大学 | Strain of Dongxiang wild rice endophytic fungus for producing GAMG (glycyrrhetinic acid mono-glucuronide) by efficiently converting glycyrrhizic acid and application thereof |
| CN109439697A (en) * | 2018-10-25 | 2019-03-08 | 黑龙江大学 | The method for producing high-efficiency antioxidant active material using microbial fermentation |
| CN111485012A (en) * | 2019-01-25 | 2020-08-04 | 江西科技师范大学 | Method for preparing glycyrrhetinic acid monoglucuronide by liquorice fermentation |
| CN115044520A (en) * | 2022-08-12 | 2022-09-13 | 北京百奥茵诺生物科技有限公司 | Alteromonas and method for producing glycyrrhetinic acid monoglucuronide by using alteromonas |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106047839A (en) * | 2016-04-19 | 2016-10-26 | 北京理工大学 | Fermentation medium and method for improving activity of beta-glucuronidase produced by fungi |
| CN106701604A (en) * | 2017-03-24 | 2017-05-24 | 江西科技师范大学 | Strain of Dongxiang wild rice endophytic fungus for producing GAMG (glycyrrhetinic acid mono-glucuronide) by efficiently converting glycyrrhizic acid and application thereof |
| 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 |
| CN109439697A (en) * | 2018-10-25 | 2019-03-08 | 黑龙江大学 | The method for producing high-efficiency antioxidant active material using microbial fermentation |
| CN109439697B (en) * | 2018-10-25 | 2021-08-17 | 黑龙江大学 | Method for producing high-efficiency antioxidant active substance by utilizing microbial fermentation |
| CN111485012A (en) * | 2019-01-25 | 2020-08-04 | 江西科技师范大学 | Method for preparing glycyrrhetinic acid monoglucuronide by liquorice fermentation |
| CN111485012B (en) * | 2019-01-25 | 2023-06-09 | 江西科技师范大学 | Method for preparing glycyrrhetinic acid monoglucuronide by liquorice fermentation |
| CN115044520A (en) * | 2022-08-12 | 2022-09-13 | 北京百奥茵诺生物科技有限公司 | Alteromonas and method for producing glycyrrhetinic acid monoglucuronide by using alteromonas |
| CN115044520B (en) * | 2022-08-12 | 2022-11-15 | 北京百奥茵诺生物科技有限公司 | Alteromonas and method for producing glycyrrhetinic acid monoglucuronide by using alteromonas |
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