CN102676404A - Endophytic fungi capable of improving content of isofraxidin in Radix Acanthopanacis Semticosi through fermenting Radix Acanthopanacis Semticosi - Google Patents
Endophytic fungi capable of improving content of isofraxidin in Radix Acanthopanacis Semticosi through fermenting Radix Acanthopanacis Semticosi Download PDFInfo
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Abstract
The invention discloses to endophytic fungi capable of improving a content of isofraxidin in Radix Acanthopanacis Semticosi through fermenting the Radix Acanthopanacis Semticosi, and relates to endophytic fungi. The endophytic fungi capable of improving the content of the isofraxidin in the Radix Acanthopanacis Semticosi through fermenting the Radix Acanthopanacis Semticosi is Aspergillus ochraceus AJ14, which is preserved in China Center for Type Culture Collection with a conversation number of CCTCC No: M 2012042, a conversation address of Wuhan University, Wuhan, and a conversation data of February 29, 2012. According to the invention, the Radix Acanthopanacis Semticosi is fermented by the aspergillus ochraceus AJ14, the contents of a main active constituent isofraxidin of the Radix Acanthopanacis Semticosi before and after fermentation of the Radix Acanthopanacis Semticosi are determined by an HPLC (High Performance Liquid Chromatography) method, the content of main active constituent isofraxidin of the Radix Acanthopanacis Semticosi can be remarkably improved to a certain degree by the endophytic fungi AJ14 of the Radix Acanthopanacis Semticosi, and the endophytic fungi provided by the invention has guidance significance for effectively producing the isofraxidin component in the Radix Acanthopanacis Semticosi by fermenting the Radix Acanthopanacis Semticosi through the endophytic fungi AJ14 and saving medicine sources.
Description
Technical field
The present invention relates to a strain endogenetic fungus.
Background technology
Radix Et Caulis Acanthopanacis Senticosi is dry root and rhizome or the stem of Araliaceae Radix Et Caulis Acanthopanacis Senticosi (Acanthopanax senticosus (rupr.et Maxim.) Harms), is not only to have eaten but also the medicine functional health-care food.The activeconstituents of Radix Et Caulis Acanthopanacis Senticosi mainly contains: isofraxidin, Syringin, polysaccharide etc., have antifatigue, and anti-inflammatory, anti-oxidant, immunomodulatory, step-down, effect such as hypoglycemic grade is widely used in medicine and food service industry at present.Because the Radix Et Caulis Acanthopanacis Senticosi demand goes up, area of woods reduces and excavating blindly gradually, makes the Radix Et Caulis Acanthopanacis Senticosi resource face test, and Radix Et Caulis Acanthopanacis Senticosi is the medicinal plant in imminent danger of two types of focused protections of country at present.
Fermentation technique can make some activeconstituents generation increase and decrease of food, and some activeconstituents in the protection food is saved resource, for the acquisition of food, pharmaceutical active ingredient provides novel method.
Summary of the invention
The invention provides the endogenetic fungus that strain fermentation Radix Et Caulis Acanthopanacis Senticosi improves isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi.
The present invention's Radix Et Caulis Acanthopanacis Senticosi that ferments improves the endogenetic fungus of isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi; It is Aspergillus ochraceus (Aspergillus ochraceus) AJ14; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012042, 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 3～5cm, and radial rill is arranged slightly, tool concentricity, white mycelium; Conidium face ochre yellow, quality velvet shape, a small amount of colourless transudate of tool, reverse side tawny; Conidial head is spherical, and mitogenetic falx stem wall is coarse, and the top capsule is spherical, diameter 25～45 μ m; The surface all can be educated, and conidial fructification is double-deck, and the metulae size is 5～10 μ m * 3～4 μ m, and bottle stalk size is 8～11 μ m * 3～4 μ m; Conidium is spherical, diameter 2.5～3.5 μ m.
The present invention's Radix Et Caulis Acanthopanacis Senticosi that ferments improves the endogenetic fungus of isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi; It is Aspergillus ochraceus (Aspergillus ochraceus) AJ14; 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 similarity that the ITS sequence of AJ14 bacterial strain and Aspergillus ochraceus belong to bacteria strain has all reached more than 98%; Combining form is learned observations, confirms that the AJ14 bacterial strain is Aspergillus ochraceus (Aspergillus ochraceus).
The present invention's Radix Et Caulis Acanthopanacis Senticosi that ferments improves the endogenetic fungus of isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi; It is Aspergillus ochraceus (Aspergillus ochraceus) AJ14; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012042, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
The present invention adopts separation from the endogenetic fungus AJ14 of Radix Et Caulis Acanthopanacis Senticosi fermentation Radix Et Caulis Acanthopanacis Senticosi; With main active ingredient isofraxidin in the Radix Et Caulis Acanthopanacis Senticosi is index; The relatively dynamic change of isofraxidin content before and after its fermentation; Be intended to improve the content of main active ingredient isofraxidin, for efficiently utilizing Radix Et Caulis Acanthopanacis Senticosi, saving medicine source, development of new functional health-care food provide foundation.Correlative study about adopting endogenetic fungus AJ14 fermentation Radix Et Caulis Acanthopanacis Senticosi does not appear in the newspapers at present.
Radix Et Caulis Acanthopanacis Senticosi endogenetic fungus AJ14 of the present invention separates the stem from healthy wild Radix Et Caulis Acanthopanacis Senticosi.This bacterial strain has spore; Its diameter is 25 μ m; Be accredited as Aspergillus ochraceus (Aspergillus ochraceus) through morphological observation and 18S rDNA sequential analysis; It can make, and isofraxidin content significantly improves in the Radix Et Caulis Acanthopanacis Senticosi, therefore in the raw material production that with the acquisition isofraxidin is purpose, can select Radix Et Caulis Acanthopanacis Senticosi endogenetic fungus AJ14 fermentation Radix Et Caulis Acanthopanacis Senticosi to improve yield, saves the medicine source.
The present invention's Radix Et Caulis Acanthopanacis Senticosi that ferments improves the endogenetic fungus of isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi; It is Aspergillus ochraceus (Aspergillus ochraceus) AJ14; Adopt AJ14 that Radix Et Caulis Acanthopanacis Senticosi is fermented; The HPLC method is measured the content of the different piperazine pyridine of Radix Et Caulis Acanthopanacis Senticosi fermentation front and back Radix Et Caulis Acanthopanacis Senticosi main active ingredient; Its result shows that Radix Et Caulis Acanthopanacis Senticosi endogenetic fungus AJ14 can make the different piperazine pyridine of Radix Et Caulis Acanthopanacis Senticosi main active ingredient be able to a certain extent significantly improve, and explains that the present invention has directive significance to adopting isofraxidin composition, saving medicine source in the endogenetic fungus AJ14 fermentation Radix Et Caulis Acanthopanacis Senticosi High-efficient Production Radix Et Caulis Acanthopanacis Senticosi.
Description of drawings
Fig. 1 is the colonial morphology figure of Aspergillus ochraceus in the embodiment one (Aspergillus ochraceus) AJ14;
Fig. 2 is the spore micro-structure diagram of Aspergillus ochraceus in the embodiment one (Aspergillus ochraceus) AJ14;
Fig. 3 is the electrophorogram of Aspergillus ochraceus in the embodiment one (Aspergillus ochraceus) AJ14 genomic dna, and wherein the M swimming lane is represented Marker;
Fig. 4 is the systematic evolution tree of Aspergillus ochraceus in the embodiment one (Aspergillus ochraceus) AJ14;
Fig. 5 is the HPLC color atlas of different piperazine pyridine reference substance in the embodiment one, wherein the different piperazine pyridine of 1 expression;
Fig. 6 is the HPLC color atlas of Caulis Et Caulis Acanthopanacis Senticosi in the embodiment one, wherein the different piperazine pyridine of 1 expression;
Fig. 7 is the HPLC color atlas of Caulis Et Caulis Acanthopanacis Senticosi after the AJ14 fermentation in the embodiment one, the wherein different piperazine pyridine of 1 expression.
Embodiment
Embodiment one: this embodiment fermentation Radix Et Caulis Acanthopanacis Senticosi improves the endogenetic fungus of isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi; It is Aspergillus ochraceus (Aspergillus ochraceus) AJ14; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012042, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
Aspergillus ochraceus in this embodiment (Aspergillus ochraceus) AJ14, it is after cultivating 7d on the PDA solid medium, and colony diameter is 3～5cm, and radial rill is arranged slightly; Tool concentricity, white mycelium, conidium face ochre yellow, quality velvet shape; The a small amount of colourless transudate of tool, the reverse side tawny, conidial head is spherical, and mitogenetic falx stem wall is coarse; The top capsule is spherical, diameter 25～45 μ m, and the surface all can be educated; Conidial fructification is double-deck, and the metulae size is 5～10 μ m * 3～4 μ m, and bottle stalk size is 8～11 μ m * 3～4 μ m; Conidium is spherical, diameter 2.5～3.5 μ m.
Aspergillus ochraceus in this embodiment (Aspergillus ochraceus) AJ14, its growth temperature is 28 ℃, growth pH value is a nature.
The fermentation Radix Et Caulis Acanthopanacis Senticosi improves the endogenetic fungus of isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi in this embodiment; It is Aspergillus ochraceus (Aspergillus ochraceus) AJ14; This strains separation picks up from area, cap mountain, Heilongjiang Province, 3 years plant ages from the stem of healthy wild Radix Et Caulis Acanthopanacis Senticosi; Robust plant, no disease and pest.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 the stem of healthy wild Radix Et Caulis Acanthopanacis Senticosi and rinse the back well with the aseptic filter paper suck dry moisture with tap water; Be cut into the 0.5cm segment, the stem with Radix Et Caulis Acanthopanacis Senticosi in aseptic super clean bench carries out surface sterilization as follows: 75% alcohol-pickled 3～5min-2% Youxiaolin soaks the 15min-aseptic water washing 4 times.With the aseptic operation cutter sample is cut from the centre; Place yam solid separation culture medium and yam liquid separation culture medium respectively; Cultivate in 28 ℃ of constant incubators and the shaking table, treat that its edge and top grow mycelia after, in time picking colony is transferred in the corresponding substratum; Get the sterilized water coating culture plate of last flushing Caulis Et Caulis Acanthopanacis Senticosi or get an amount of this washing fluid and pour in the yam liquid separation culture medium and compare, the experiment material after will sterilizing is in addition rolled on culture plate and is compared in a week, under identical condition, cultivates.
Wherein the every L of yam solid separation culture medium is made up of the yam of 200g, the glucose of 20g, the agar of 20g and the zero(ppm) water of surplus, and the pH value is 7.0～7.2,121 ℃ of following autoclaving 30min;
The every L of yam liquid separation culture medium is made up of the yam of 200g, the glucose of 20g and the zero(ppm) water 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 5mL 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 Caulis Et Caulis Acanthopanacis Senticosi, isolate endogenetic fungus AJ14; And all not having any bacterium in contrast flat board and the control liquid substratum in the negative control grows; Repeatedly repeat all so, prove that the bacterium of being assigned to is a plant endogenesis epiphyte, rather than the epiphyte on surface.The endogenetic fungus that filters out carries out morphology according to " classification of fungi ", " fungi identification handbook " and " Dendrochium diagram " to be identified; It is very close that the colony characteristics of bacterial strain and spore shape and Aspergillus ochraceus belong to the bacterium characteristic; Colonial morphology is as shown in Figure 1, and the spore microstructure is as shown in Figure 2;
Molecular Identification:
Endogenetic fungus AJ14 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 adopts Shanghai to give birth to worker UNIQ-10 pillar fungal gene group extraction agent box then and extracts genomic dna, carries out the segmental pcr amplification of purpose (the gel imaging result is as shown in Figure 3) then; Near 500bp (base sequence is seen SEQ ID NO:1), obtain an amplified fragments, prove successfully from the AJ14 bacterial strain, to extract its genomic dna; The PCR product that will contain target stripe all carries out point sample again; Agarose gel electrophoresis with 2% downcuts the purpose band with scalper under uv lamp, reclaims test kit with DNA glue and reclaims; The preparation competent escherichia coli cell; The PCR product with after pMD 18-T carrier is connected, is connected product and joins 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 MEGA5.03 constructing system of corresponding kind representative strain to grow according to the ultimate principle of molecular systematics research and set, confirm the classification position of aimed strain.
The ITS sequence that records 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 (as shown in Figure 4); The similarity that the ITS sequence of AJ14 bacterial strain and Aspergillus ochraceus belong to bacteria strain has all reached more than 98%, and combining form is learned observations, confirms that the AJ14 bacterial strain is Aspergillus ochraceus (Aspergillus ochraceus).
The HPLC method is measured the dynamic change of endogenetic fungus AJ14 to isofraxidin before and after the Radix Et Caulis Acanthopanacis Senticosi fermentation:
The preparation of 1 trial-product and reference substance solution
(1) chromatographic condition:
Chromatographic column: VenusiL XBP-C
18Post (4.6mm * 250mm, 5 μ m);
Moving phase: 0.1% phosphoric acid-acetonitrile (80: 20);
Flow velocity: 1mLmin
-1
Detect wavelength: 343nm;
Column temperature: 25 ℃;
Sample size: 10 μ L.
(2) preparation of reference substance solution
Precision takes by weighing the isofraxidin standard substance, and each is an amount of, is that solvent is processed the reference substance storing solution that mass concentration is 1.0mg/mL respectively with methyl alcohol.Accurately measure reference substance storing solution 1mL, process the reference substance solution that mass concentration is 0.25mg/mL.
(3) need testing solution preparation
The preparation of fermented sample: get the exsiccant Radix Et Caulis Acanthopanacis Senticosi and pulverize, cross 60 mesh sieves.Accurately take by weighing the Radix Et Caulis Acanthopanacis Senticosi 15g after the pulverizing, place Erlenmeyer flask, add water, after the sealing,, take out, as substrate in 121 ℃ of autoclaving 30min according to 1: 5 solid-liquid ratio.
With endogenetic fungus AJ14 activation in the PDA substratum, in the endogenetic fungus AJ14 that activation is good, add a certain amount of sterilized water, process 1 * 10
7The bacteria suspension of CFU/mL.The 5mL bacteria suspension is joined in the substrate after the sterilization; Other gets substrate and adds the 5mL sterilized water as the blank sample, and each 10 parts of parallel sample are put into shaking table in 37 ℃ with all samples, cultivate 15 days, take out, and freeze-drying, subsequent use.
The preparation of crude drug reference substance solution: get the exsiccant Caulis Et Caulis Acanthopanacis Senticosi, pulverize, cross 40 mesh sieves.Precision takes by weighing 3 parts of each 1.0g of Manyprickle Acanthopanax Root, accurately adds 10mL methyl alcohol, weighs, and supersound extraction 60min takes out, and puts coldly, supplies the solvent that subtracts mistake with methyl alcohol, with behind the 0.45 μ m filtering with microporous membrane as reference substance solution.
The preparation of fermented sample need testing solution: precision takes by weighing the Caulis Et Caulis Acanthopanacis Senticosi sample 1.0g after endogenetic fungus AJ14 fermentation; The accurate 10mL methyl alcohol that adds, supersound extraction 60min takes out; Put cold; Supply the solvent that subtracts mistake with methyl alcohol, with 0.45 μ m filtering with microporous membrane, i.e. Radix Et Caulis Acanthopanacis Senticosi fermented sample need testing solution.
The preparation of blank article solution: get blank sample 1.0g, the accurate 10mL methyl alcohol that adds, supersound extraction 60min takes out, and puts coldly, supplies the solvent that subtracts mistake with methyl alcohol, with 0.45 μ m filtering with microporous membrane, is blank article solution.
The preparation of fermented liquid reference substance solution: precision is measured each 100mL of Radix Et Caulis Acanthopanacis Senticosi endogenetic fungus AJ14 fermented liquid, and rotary evaporation is concentrated into 10mL, with 0.45 μ m filtering with microporous membrane, is the fermented liquid reference substance solution.
(3) sample determination is under " (1) " chromatographic condition, and precision is drawn 10 μ L (three times are parallel) reference substance solution and each need testing solution respectively, sample introduction, and the record peak area is by external standard method calculating content.
2 detected results
Under chromatographic condition, need testing solution is consistent with the chromatographic peak of isofraxidin reference substance corresponding position, and chromatographic peak has obtained confirming preferably through the reference substance addition method; The result sees Fig. 5; Fig. 6, Fig. 7 and table 1, experiment shows; After endogenetic fungus AJ14 fermentation, the content of different piperazine pyridine has raising extremely significantly in the Caulis Et Caulis Acanthopanacis Senticosi.
The assay result of different piperazine pyridine in table 1 Caulis Et Caulis Acanthopanacis Senticosi
Annotate: compare with crude drug
*P＜0.05,
*P＜0.01; Compare with the blank group
△P＜0.05,
△ △P＜0.01.
Conclusion: adopt Radix Et Caulis Acanthopanacis Senticosi endogenetic fungus AJ14 that Radix Et Caulis Acanthopanacis Senticosi is fermented; The HPLC method is measured the content of the different piperazine pyridine of Radix Et Caulis Acanthopanacis Senticosi fermentation front and back Radix Et Caulis Acanthopanacis Senticosi main active ingredient; Its result shows; Radix Et Caulis Acanthopanacis Senticosi endogenetic fungus AJ14 can make the different piperazine pyridine of Radix Et Caulis Acanthopanacis Senticosi main active ingredient be able to a certain extent significantly improve, and explains that the present invention has directive significance to adopting isofraxidin composition, saving medicine source in the endogenetic fungus AJ14 fermentation Radix Et Caulis Acanthopanacis Senticosi High-efficient Production Radix Et Caulis Acanthopanacis Senticosi.
Claims (1)
1. strain fermentation Radix Et Caulis Acanthopanacis Senticosi improves the endogenetic fungus of isofraxidin content in the Radix Et Caulis Acanthopanacis Senticosi; It is characterized in that it is Aspergillus ochraceus (Aspergillus ochraceus) AJ14; In China's typical culture collection center preservation; Deposit number is CCTCC No:M 2012042, and the preservation address is a Wuhan City Wuhan University, and preservation date is on February 29th, 2012.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103720733A (en) * | 2014-01-23 | 2014-04-16 | 程钰翔 | Preparation method of manyprickle acanthopanax root extract |
| CN108513990A (en) * | 2018-03-21 | 2018-09-11 | 黑龙江中医药大学 | A method of for promoting wilsonii seedling growth |
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| WO2008154750A1 (en) * | 2007-06-21 | 2008-12-24 | Innodia Inc. | Conversion of isoleucine to 4-hydroxyisoleucine by microorganisms |
| CN101851219A (en) * | 2010-05-27 | 2010-10-06 | 东北林业大学 | Method for preparing isofraxidin from acanthopanax senticosus rhizomes |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008154750A1 (en) * | 2007-06-21 | 2008-12-24 | Innodia Inc. | Conversion of isoleucine to 4-hydroxyisoleucine by microorganisms |
| CN101851219A (en) * | 2010-05-27 | 2010-10-06 | 东北林业大学 | Method for preparing isofraxidin from acanthopanax senticosus rhizomes |
Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103720733A (en) * | 2014-01-23 | 2014-04-16 | 程钰翔 | Preparation method of manyprickle acanthopanax root extract |
| CN108513990A (en) * | 2018-03-21 | 2018-09-11 | 黑龙江中医药大学 | A method of for promoting wilsonii seedling growth |
| CN108513990B (en) * | 2018-03-21 | 2019-05-28 | 黑龙江中医药大学 | A method of for promoting wilsonii seedling growth |
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