CN102643756A - Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice - Google Patents
Endophytic fungus for improving content of glycyrrhetinic acid by fermenting liquorice Download PDFInfo
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- CN102643756A CN102643756A CN2012101262869A CN201210126286A CN102643756A CN 102643756 A CN102643756 A CN 102643756A CN 2012101262869 A CN2012101262869 A CN 2012101262869A CN 201210126286 A CN201210126286 A CN 201210126286A CN 102643756 A CN102643756 A CN 102643756A
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Abstract
The invention discloses an endophytic fungus for improving the content of glycyrrhetinic acid by fermenting liquorice and relates to an endophytic fungus. The endophytic fungus is aspergillus versicolor RE4 and is preserved at the China Center for Type Culture Collection, wherein the preservation number is CCTCC No: M2012045; the preservation address is Wuhan University in Wuhan; and the preservation date is Feb. 29th, 2012. The endophytic fungus is fermented by taking the liquorice as a substrate, so that the content of glycyrrhetinic acid in the liquorice can be obviously improved; the glycyrrhetinic acid is a classical anti-inflammatory drug, is widely applied in the fields such as medical cosmetics and the like and also has functions of cancer prevention and cancer resistance. The invention has guiding significance in producing the glycyrrhetinic acid by fermenting the liquorice by adopting the endophytic fungus RE4.
Description
Technical field
The present invention relates to a strain endogenetic fungus.
Background technology
Mikrobe particularly fungi has the ability of very strong decomposition and inversion material; And can produce abundant secondary metabolite, through microbial fermentation herbal medicine and general physical or chemical process method mutually specific energy change the property of medicine significantly, the raising curative effect; Reduce toxic side effect, enlarge indication.Utilize the endogenetic fungus in the plant that Chinese medicine is fermented; Because some material of Chinese medicine possibly have promotion or restraining effect to microbial growth and metabolism; Generation to activeconstituents also has promotion or restraining effect; Mikrobe also possibly change the pathways metabolism of self in the particular surroundings of Chinese medicine; Thereby form new activeconstituents or change the mutual ratio of each activeconstituents, therefore utilize the endogenetic fungus that from medicinal plant, is separated to produce the research tendency that active substances has become current scholar next life.
Summary of the invention
The invention provides strain fermentation Radix Glycyrrhizae and improve the endogenetic fungus of glycyrrhetinic acid content.
The present invention Radix Glycyrrhizae of fermenting is improved the endogenetic fungus of glycyrrhetinic acid content; It is aspergillus versicolor (Aspergillus versicolor) RE4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012045, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012; It is after cultivating 7d on the PDA solid medium, and colony diameter is 12～14cm, and bacterium colony is a white, fades to greyish-green, and matrix is pale brown look; The mycelia comparatively dense is panniform, and colony edge is neat, is close to substratum; Produce conidium, conidial head is spherical in shape, and conidium Dan Sheng is unicellular.
The present invention Radix Glycyrrhizae of fermenting is improved the endogenetic fungus of glycyrrhetinic acid content; It is aspergillus versicolor (Aspergillus versicolor) RE4; Its ITS sequence is committed to the NCBI webpage, through Blast search and the high sequence of resulting sequence similarity, and through MEGA5.03 software building systematic evolution tree; The ITS sequence of RE4 bacterial strain and Aspergillus bacterium (Aspergillus versicolor; Numbering JN545818.1) similarity of bacterial strain has all reached more than 98%, and combining form is learned observations, confirms that endogenetic fungus RE4 is aspergillus versicolor (Aspergillus versicolor).
The present invention Radix Glycyrrhizae of fermenting is improved the endogenetic fungus of glycyrrhetinic acid content; It is aspergillus versicolor (Aspergillus versicolor) RE4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012045, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
The present invention Radix Glycyrrhizae of fermenting is improved the endogenetic fungus of glycyrrhetinic acid content, and it be aspergillus versicolor (Aspergillus versicolor) RE4, can be that substrate ferments to it with the Radix Glycyrrhizae, main active ingredient---the content of glycyrrhetinic acid in the raising Radix Glycyrrhizae.
The present invention utilizes the endogenetic fungus RE4 of microbiological pharmacy means extraction separation Radix Glycyrrhizae healthy plant; With the Radix Glycyrrhizae is that substrate ferments; Adopting the HPLC method is the content of glycyrrhetinic acid behind the analysis means mensuration Radix Glycyrrhizae endogenetic fungus RE4 fermentation Radix Glycyrrhizae, at last endogenetic fungus RE4 bacterial strain is identified its kind through morphological observation and 18S rDNA sequential analysis.
This strain among the present invention can improve the endogenetic fungus RE4 of glycyrrhetinic acid through the fermentation Radix Glycyrrhizae; It is aspergillus versicolor (Aspergillus versicolor); It is that substrate ferments to it glycyrrhetinic acid content in Radix Glycyrrhizae is significantly raise with the Radix Glycyrrhizae; Glycyrrhetinic acid is classical anti-inflammatory drug, and widespread use in fields such as medical makeup at present also has anti-cancer and antitumous effect; The present invention has directive significance to adopting endogenetic fungus RE4 fermentation Radix Glycyrrhizae to produce glycyrrhetinic acid.
Description of drawings
Fig. 1 is the spore micro-structure diagram of aspergillus versicolor in the embodiment one (Aspergillus versicolor) RE4;
Fig. 2 is the electrophorogram of the pcr amplification of aspergillus versicolor in the embodiment one (Aspergillus versicolor) RE4, and wherein the M swimming lane is represented Marker, and 1 swimming lane is represented RE4;
Fig. 3 is the phyletic evolution tree graph of aspergillus versicolor in the embodiment one (Aspergillus versicolor) RE4;
Fig. 4 is the HPLC color atlas of glycyrrhetinic acid reference substance in the embodiment one, wherein 1 expression glycyrrhetinic acid;
Fig. 5 is the HPLC color atlas of Radix Glycyrrhizae in the embodiment one, wherein 1 expression glycyrrhetinic acid;
Fig. 6 is the HPLC color atlas that Radix Glycyrrhizae adds glycyrrhetinic acid behind the reference substance in the embodiment one, and wherein 1 expression adds glycyrrhetinic acid behind the reference substance;
Fig. 7 is the HPLC color atlas of glycyrrhetinic acid behind the RE4 fermentation Radix Glycyrrhizae in the embodiment one, and wherein 1 expression adds glycyrrhetinic acid behind the reference substance.
Embodiment
Embodiment one: this embodiment fermentation Radix Glycyrrhizae is improved the endogenetic fungus of glycyrrhetinic acid content; It is aspergillus versicolor (Aspergillus versicolor) RE4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012045, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
Aspergillus versicolor in this embodiment (Aspergillus versicolor) RE4, it is after cultivating 7d on the PDA solid medium, and colony diameter is 12～14cm, and bacterium colony is a white; Fade to greyish-green, matrix is pale brown look, and the mycelia comparatively dense is panniform; Colony edge is neat, is close to substratum, produces conidium; Conidial head is spherical in shape, and conidium Dan Sheng is unicellular.
Aspergillus versicolor in this embodiment (Aspergillus versicolor) RE4, its growth temperature is 28 ℃, growth pH value nature.
Aspergillus versicolor in this embodiment (Aspergillus versicolor) RE4, this strains separation picks up from Daqing Area from healthy wild Radix Glycyrrhizae rhizome; 3 years plant ages; Robust plant, no disease and pest, its sample is existing in bio-pharmaceuticals specialized laboratory of life science institute of Heilongjiang University; It carries out separation and Culture according to the following steps:
Choose healthy wild Radix Glycyrrhizae rhizome and rinse the back well with the aseptic filter paper suck dry moisture with tap water, be cut into the 1cm segment and in aseptic super clean bench, wild Radix Glycyrrhizae rhizome carried out surface sterilization as follows: 75% alcohol-pickled 1min-2% Youxiaolin soaks the 15min-aseptic water washing 3 times; With the aseptic operation cutter wild Radix Glycyrrhizae rhizome is cut from the centre; Place 5% licorice extract yam solid separation culture medium and 5% licorice extract yam liquid separation culture medium respectively; In 28 ℃ of water bath with thermostatic control shaking tables and constant incubator; Cultivate 3～5d, treat to grow thalline around the experiment material, the picking thalline changes flat board (the 5% licorice extract yam solid separation culture medium) separation and purification of repeatedly ruling over to; Bacterial strain behind the purifying is placed the inclined-plane solid medium, and 4 ℃ of preservations are subsequent use;
The every L of 5% licorice extract yam solid separation culture medium is made up of the yam of 200g, the glucose of 20g, the agar of 20g and 5% licorice extract of surplus, and the pH value is 7.0～7.2,121 ℃ of following autoclaving 30min;
The every L of 5% licorice extract yam liquid separation culture medium is made up of the yam of 200g, the glucose of 20g and 5% licorice extract of surplus, and the pH value is 7.0～7.2,121 ℃ of following autoclaving 30min;
The inclined-plane solid medium is loaded in the test tube for getting 5mL5% licorice extract yam solid separation culture medium, behind 121 ℃ of autoclaving 30min, is paved into the inclined-plane cooling, in order to preserving bacterial classification.
Result: from wild Radix Glycyrrhizae rhizome, isolate endogenetic fungus RE4; Carrying out morphology according to " classification of fungi ", " fungi identification handbook " and " Dendrochium diagram " identifies; Its colony characteristics and spore shape are very close with Aspergillus bacterium characteristic, and the spore microstructure is as shown in Figure 1;
Molecular Identification: endogenetic fungus RE4 fermented liquid suction filtration gets mycelium with 25% ethanol rinsing 2 times, the deionized water rinsing of sterilization 2 times, the centrifugal supernatant that goes; Carry out the segmental pcr amplification of purpose after extracting total DNA, the PCR product that will contain target stripe all carries out point sample again, the agarose gel electrophoresis with 2%; Under uv lamp, the purpose band is downcut with scalper; Reclaim test kit with DNA glue and reclaim, preparation competence Bacillus coli cells, with the PCR product with after the pMD18-T carrier is connected; Connect product and join in the competent cell, carry out bacterium colony PCR checking; The PCR that picking list bacterium colony carries out the intestinal bacteria transformant detects, prove the purpose fragment successfully transforms get in the competent cell after, will clone positive strain and serve extra large living worker's biotechnology Services Co., Ltd and check order; The nucleotides sequence of being measured is listed in the ncbi database Application of B LAST and analyzes and carry out homology relatively; And select the 18S rDNA sequence application software CLUSTALX 1.83 of corresponding kind representative strain and MEGA5.03 to grow tree with neighbour's combined techniques (Neighbor-joining) constructing system according to the ultimate principle of molecular biology research; Bootstrap test value >=50% (1000 repeat) is confirmed the classification position of aimed strain;
Endogenetic fungus RE4 genome is after Wyler's process extracts; The gel imaging result is as shown in Figure 2 after adopting pcr amplification; Near 500bp, obtain an amplified fragments (base sequence is seen SEQ ID NO:1), prove successfully from endogenetic fungus RE4 bacterial strain, to extract its genomic dna;
ITS sequence according to endogenetic fungus RE4 is committed to the NCBI webpage; Through Blast search and the high sequence of resulting sequence similarity; And through MEGA5.03 software building systematic evolution tree (see figure 3), the similarity of the ITS sequence of RE4 bacterial strain and Aspergillus bacterium (Aspergillus versicolor, numbering JN545818.1) bacterial strain has all reached more than 98%; Combining form is learned observations, confirms that endogenetic fungus RE4 is that aspergillus versicolor belongs to bacterium (Aspergillus versicolor).
Aspergillus versicolor in this embodiment (Aspergillus versicolor) RE4; The experimental result that its fermentation Radix Glycyrrhizae is improved glycyrrhetinic acid is as shown in table 1; Endogenetic fungus RE4 fermentation Radix Glycyrrhizae is improved the glycyrrhetinic acid experimental result and shows, endogenetic fungus RE4 can significantly raise glycyrrhetinic acid content wherein through the fermentation Radix Glycyrrhizae.
Table 1
Annotate: compare * P＜0.05 with the crude drug group, * * P＜0.01; Compare △ P＜0.05 with negative control group, △ △ P＜0.01; The no glycyrrhetinic acid of PDA contrast produces.
Aspergillus versicolor in this embodiment (Aspergillus versicolor) RE4, the detection that its fermentation Radix Glycyrrhizae is improved glycyrrhetinic acid content is following:
Get dry licorice, pulverize the back with kibbler and cross 40 mesh sieves, accurately take by weighing the 6g licorice powder, put into the 100mL triangular flask, again to the zero(ppm) water that wherein adds 30mL, after the sealing, 121 ℃ of autoclaving 30min.Take out, as substrate.
To adding sterilized water in the activatory endogenetic fungus RE4 bacterial strain test tube, process 1 * 10
6The CFU/mL bacteria suspension is got this bacteria suspension 5mL and is joined in the substrate; Other gets sterilized water that substrate adds equivalent as blank, and in 28 ℃, the 120r/min shaking table was cultivated 14 days with above-mentioned each sample for preparing.Take out, freeze-drying is the Radix Glycyrrhizae fermented sample, and is subsequent use.Each sample is done 6 repetitions.
Precision takes by weighing Radix Glycyrrhizae fermented sample 1g, accurately adds 10mL chromatogram methyl alcohol, weighs, and supersound extraction 2h takes out, and puts coldly, supplies weight with chromatogram methyl alcohol, filters, and gets subsequent filtrate 4mL, and constant volume in the 10mL volumetric flask is to be measured.
Simultaneously precision takes by weighing Radix Glycyrrhizae crude drug 1g, accurately adds the chromatogram methyl alcohol of 10mL, weighs, and supersound extraction 2h takes out, and puts coldly, supplies weight with chromatogram methyl alcohol, gets subsequent filtrate 4mL, and constant volume in the 10mL volumetric flask is to be measured as the crude drug contrast.
Glycyrrhetinic acid Determination on content before and after the Radix Glycyrrhizae fermentation:
Chromatographic condition
Chromatographic column: VenusiL XBP-C
18Post (4.6mm * 250mm, 5 μ m);
Moving phase: methanol-water-Glacial acetic acid min. 99.5 (82: 17: 1);
Flow velocity: 1mLmin
-1
Detect wavelength: 254nm;
Column temperature: 25 ℃;
Sample size: 10 μ L
Under chromatographic condition; The prepared trial-product of Radix Glycyrrhizae endogenetic fungus RE4 fermentation Radix Glycyrrhizae is consistent with the chromatographic peak of glycyrrhetinic acid reference substance corresponding position; And chromatographic peak has obtained confirming preferably through the reference substance addition method; The result sees Fig. 4,5,6 and 7, conclusion: the fermentation Radix Glycyrrhizae is improved the endogenetic fungus of glycyrrhetinic acid content, i.e. aspergillus versicolor (Aspergillus versicolor) RE4; It is that substrate ferments glycyrrhetinic acid content is significantly raise with the Radix Glycyrrhizae, and this embodiment has directive significance to adopting Radix Glycyrrhizae endogenetic fungus RE4 to produce glycyrrhetinic acid.
Claims (1)
1. strain fermentation Radix Glycyrrhizae is improved the endogenetic fungus of glycyrrhetinic acid content; It is characterized in that it is aspergillus versicolor (Aspergillus versicolor) RE4; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012045, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774901A (en) * | 2014-01-15 | 2015-07-15 | 上海医药工业研究院 | Method for preparing glycyrrhetinic acid and its derivatives by microbial transformation |
| CN104805169A (en) * | 2014-01-27 | 2015-07-29 | 上海医药工业研究院 | Method for producing glycyrrhetinic acid through microbial transformation, and medium 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 |
| CN109504647A (en) * | 2019-01-04 | 2019-03-22 | 湖南农业大学 | A kind of cultural method of aspergillus versicolor HY12 bacterial strain |
| CN109628325A (en) * | 2019-01-04 | 2019-04-16 | 湖南农业大学 | A kind of aspergillus versicolor HY12 bacterial strain |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774901A (en) * | 2014-01-15 | 2015-07-15 | 上海医药工业研究院 | Method for preparing glycyrrhetinic acid and its derivatives by microbial transformation |
| CN104774901B (en) * | 2014-01-15 | 2018-05-04 | 上海医药工业研究院 | A kind of method of microorganism conversion generation enoxolone and its derivative |
| CN104805169A (en) * | 2014-01-27 | 2015-07-29 | 上海医药工业研究院 | Method for producing glycyrrhetinic acid through microbial transformation, and medium thereof |
| CN104805169B (en) * | 2014-01-27 | 2018-04-27 | 上海医药工业研究院 | A kind of method and its culture medium of microorganism conversion production enoxolone |
| 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 |
| CN109504647A (en) * | 2019-01-04 | 2019-03-22 | 湖南农业大学 | A kind of cultural method of aspergillus versicolor HY12 bacterial strain |
| CN109628325A (en) * | 2019-01-04 | 2019-04-16 | 湖南农业大学 | A kind of aspergillus versicolor HY12 bacterial strain |
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