CN107312720A - One plant of Efficient Conversion ginsenoside Rb1 is Rd pigeonpeas endogenetic fungus and application - Google Patents
One plant of Efficient Conversion ginsenoside Rb1 is Rd pigeonpeas endogenetic fungus and application Download PDFInfo
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- CN107312720A CN107312720A CN201710115419.5A CN201710115419A CN107312720A CN 107312720 A CN107312720 A CN 107312720A CN 201710115419 A CN201710115419 A CN 201710115419A CN 107312720 A CN107312720 A CN 107312720A
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Abstract
Raw sickle-like bacteria (Fusarium proliferatum) G11 7 of pigeonpea endogenetic fungal bacterial strain layer for Rd and its application of separation method and microbe conversion ginsenoside the invention discloses ginsenoside Rb1 in one plant of Efficient Conversion pseudo-ginseng.The bacterial strain (G11 7) derives from pigeonpea plant, by solid culture, isolates and purifies and obtains.The bacterial strain can efficiently, exclusively convert ginsenoside Rb1 for Rd, and ginsenoside Rd's content after conversion is about 4 times before conversion.Layer life reaping hook bacteria strain (G11 7) of the present invention is the endogenetic fungus obtained first from pigeonpea, time needed for ginsenoside Rb1 to be converted into Rd using the bacterial strain is short, high conversion rate, green non-pollution, industrialization production is easily achieved, is had a extensive future.
Description
Technical field
The present invention relates to one plant of Efficient Conversion ginsenoside Rb1, the endogenetic fungus for Rd and its application, belong to microorganism skill
Art application field.
Technical background
Plant endogenesis epiphyte (Endophytic fungi) refers to give birth within plant tissue in a certain stage of its history of life
It is living, but do not cause the fungi of the obvious Disease symptoms of plant.Pass through " coevolution " with medicinal plant, some endogenetic fungus tools
There is the ability (Chen Lijun etc., 2006) of generation and the same or analogous bioactive substance of host plant.Endogenetic fungus has
Abundant species diversity, its secondary metabolite also very abundant.Pass through the nutrition culture to endogenetic fungus and fermentation training
A series of enzyme can be produced by supporting, and these microbe-derived enzymes can have an effect with substrate, its structure is changed,
Therefore, this kind of microorganism conversion of endogenetic fungus possesses that biomass accumulation is fast, transformation time is short, transformation efficiency is high, be easy to industry
The features such as metaplasia is produced (Guo Congliang etc., 2016).
Pseudo-ginseng [Panax notoginseng (Burk.) F.H.Chen].Mountain knee, invaluable, pseudo-ginseng are commonly called as, is Umbellales
Araliaceae, is distributed mainly on the ground such as Yunnan, Guangxi, Sichuan.Pseudo-ginseng is the traditional rare traditional Chinese medicine of China, away from the present existing 400
Applicating history for many years, at home and abroad has long enjoyed a good reputation, the laudatory title for just having " hemostasis god's medicine " from ancient times.The complicated chemical composition of pseudo-ginseng is
The basis of its good effects, the chemical composition found at present from pseudo-ginseng is main based on saponins and dencichine, and saponins is again
(Chinese Pharmacopoeias, one) such as ginsenoside, notoginsenoside and seven leaf courage saponin(es can be divided into, wherein containing with Ginsenosides Rg1 and Rb1
Measure highest.The chemical composition of the pharmacological action of pseudo-ginseng with itself is inseparable, is mainly reflected in hematological system, painstaking effort
The influence (Feng Lubing etc., 2008) of guard system, cerebrovascular system, nervous system, metabolism, immunological regulation system etc..
Ginsenoside Rd (Ginsenoside Rd), molecular formula is C48H82O18, two belonged in dammarane type ginsenoside
Alcohol type, is the rare saponin(e in pseudo-ginseng.Research finds that the pharmacological activity of ginsenoside Rd is better than other main saponin(es, and Rd is
One of principal mode absorbed after main saponin(e metabolism in enteron aisle, can protect cardiovascular and renal function, play anti-
The multiple pharmacological effect such as tumour, regulation be immune, good neuroprotective effect (Zhang Chen is also shown for central nervous system
Deng 2011).Because content of the ginsenoside Rd in plant is relatively low, cost height, mesh can be caused by directly being extracted from former vegetable drug
Compound be lost in the collection of big and raw material and limited by time and space, it is difficult to form large-scale production, and pass through interior life
The enzyme that fungal metabolite is produced carries out bioconversion to panax ginsenoside, with reaction condition it is gentle, do not destroy saponin(e structure, specially
One property is strong, high conversion rate, it is pollution-free the features such as, be expected to carry out large-scale industrial production.
The content of the invention
The invention provides the pigeonpea endogenetic fungus (Fusarium that one plant of Efficient Conversion panax ginsenoside Rb1 is Rd
Proliferatum) G11-7 and its application.Instant invention overcomes content in traditional acquisition modes is low, high energy consumption, pollution environment etc.
Shortcoming, can be achieved large-scale production, with higher application value.
The present invention provides pigeonpea endogenetic fungus and its application of one plant of Efficient Conversion panax ginsenoside Rb1 for Rd, and it is special
Levy and be the pigeonpea endogenetic fungus in PDA solid medium cultures, aerial hyphae white purplish is in royal purple sclerotium more
Color, when sclerotium when there are a lot, the lower surface of bacterium colony is presented black purple, conidium in yellowish-brown to orange, it is scattered or with viscous
Conidium, which is reunited, to be given birth to, monospore, no chlamydospore.
Pigeonpea endogenetic fungal bacterial strain of the present invention, is committed to NCBI by its gene order, is compared by BLAST, obtains gene
Accession number is KY303906, utilizes MEGA6 software building phylogenetic evolution trees, combining form observation result, it is determined that interior life
Fungus G 11-7 is the raw sickle-like bacteria of layer (Fusarium proliferatum).
Raw sickle-like bacteria (Fusarium proliferatum) G11-7 of pigeonpea endogenetic fungus layer of the present invention, by Chinese micro- life
Thing culture presevation administration committee common micro-organisms center preservation, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, deposit number is CGMCC No:13195, preservation date is on December 7th, 2016.The bacterial strain has panax ginsenoside
Rb1 Efficient Conversions are that Rd ability and its experiment show that ginsenoside Rd generates good changing effect.
Brief description of the drawings
Fig. 1 is ginsenoside Rb1, Rd structural formulas；
Fig. 2 is pigeonpea endogenetic fungus G11-7 colonial morphology figures；
Fig. 3 is the spore shape figures of pigeonpea endogenetic fungus G11-7 under an optical microscope；
Bacterium colony and transparent circle form of Fig. 4 pigeonpea endogenetic fungus G11-7 bacterial strains on cellulose-Congo red culture medium
Figure；
Fig. 5 is the phylogenetic evolution tree of the raw sickle-like bacteria of layer (Fusarium proliferatum) G11-7 bacterial strains；
Fig. 6 is that the HPLC before and after panax ginsenoside Rb1, Rd is converted through endogenetic fungus G11-7 schemes；
Embodiment
Embodiment 1：The raw sickle-like bacteria in middle level (Fusarium proliferatum) G11-7 of the present invention separation.
The raw sickle-like bacteria in present embodiment middle level (Fusarium proliferatum) G11-7, pigeonpea of the bacterial strain from health
Isolated in root, pigeonpea root picks up from Northeast Forestry University botanical garden, and plant age 3 months, its sample is existing in Northeast forestry
Key lab of the university forest plants ecological Ministry of Education；It, which is followed the steps below, is separately cultured：
(1) preparation of potato dextrose agar (PDA)：Selection is ripe, surface is smooth, without bud without the horse festered
Bell potato cleans peeling, weighs in 200g potato cuttings, boiling water boiling 30min, the potato liquid obtained after 4 layers of filtered through gauze and adds
Enter glucose 20g, agar powder 20g, addition distilled water is settled to 1L, pH naturally, 121 DEG C of autoclave sterilization 15min；
(2) healthy pigeonpea plant is taken, plant surface is cleaned with distilled water, 1min, 5% is rinsed with 75% ethanol successively
NaClO solution rinsing 5min, 10% hydrogen peroxide rinsing 3min carry out surface sterilization, with aseptic water washing 3-4 times；
(3) the pigeonpea root after sterilization, stem, leaf texture are cut into diameter about 1cm fritter with the scalpel of sterilizing respectively
Fritter tissues are inoculated in PDA solid culture primary surfaces with sterile tweezers, are positioned in insulating box, 28 DEG C of constant temperature fall by tissue
Put culture 7-10 days；
(4) mycelia grown on observation culture medium inside each plant tissue block to surrounding, using Tip Splitting picking method,
With sterile toothpick, the mycelia that the different parts edge notches directors of picking samples goes out is transferred to fresh configuration PDA culture medium flat board
On, 28 DEG C are incubated, after new bacterium colony is grown, again its Tip Splitting of picking, are transferred on PDA culture medium flat board, such as
This is repeated more than 4 times, untill the single bacterial strain purified (such as Fig. 2, Fig. 3)；
(5) fungal bacterial strain being separated to is numbered, PDA inclined-planes is inoculated in respectively, preserved under the conditions of 4 DEG C；
(6) Molecular Identification:Pigeonpea endogenetic fungus zymotic fluid obtains mycelium through four layers of filtered through gauze, extracts mycelia STb gene
The PCR amplifications of purpose fragment are carried out afterwards, send Services Co., Ltd of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to be sequenced, the nucleotide determined
Sequence carries out BLAST sequence analysis in ncbi database, and selects corresponding according to the general principle of molecular biology research
The ITS sequence of kind representative strain, application software MEGA6 is developed with neighbour's combined techniques (Neighbor-joining) constructing system
Tree, determines the classification position of aimed strain.
Embodiment 2：Raw sickle-like bacteria (the Fusarium of layer that orientation conversion panax ginsenoside Rb1 is Rd in the present invention
Proliferatum) the screening of G11-7 bacterial strains.
(1) preparation of screening and culturing medium：Cellulose-Congo red solid medium contains 0.2% sodium carboxymethylcellulose,
0.2% (NH4)2S04, 0.05%MgSO4·7H2O, 0.1%KH2P04, 1.5% agar, it is 7.0,121 DEG C of HTHPs to adjust pH
Sterilize 15min；
(2) described pigeonpea endogenetic fungal bacterial strain is taken to be inoculated into cellulose-Congo red solid medium, culture is to growing
Regular bacterium colony, covers after the Congo red solution that mass concentration is 1mg/mL, 10~15min on bacterium colony, removes Congo red solution,
NaCl solution is outwelled after adding the NaCI solution that substance withdrawl syndrome is lmol/L, 15min, now, the bacterium of cellulase is produced
Fall around to occur transparent circle, the vigor of the bigger explanation institute cellulase-producing of ratio of transparent loop diameter and colony diameter is more
It is high；
(3) test result indicates that：Pass through the screening of cellulose-Congo red solid medium, it is determined that one plant of entitled G11-7
Pigeonpea endogenetic fungus, the transparent circle of the bacterial strain and the ratio of colony diameter are 1.23 (see Fig. 4), choose G11-7 bacterial strains and carry out
Follow-up transformation experiment.
Embodiment 3：The raw sickle-like bacteria in middle level (Fusarium proliferatum) G11-7 bacterial strains orientation conversion three of the present invention
Seven ginsenoside Rb1s are Rd.
(1) actication of culture：By described raw sickle-like bacteria (Fusarium proliferatum) G11- of pigeonpea endogenetic fungus layer
7 are inoculated into culture 7-10 days in PDA solid slope culture mediums；
(2) preparation of culture medium is converted：Selection is ripe, surface is smooth, cleans and removes the peel without the potato festered without bud, weighs
200g potato cuttings, boiling water boiling 30min, the potato liquid obtained after 4 layers of filtered through gauze is settled to 1L, and pH is natural；
(3) pretreatment of the former powder of pseudo-ginseng：40 mesh sieves are crossed after pseudo-ginseng crude drug is crushed, pseudo-ginseng crude drug powder is obtained
End, the former powder of addition 2g pseudo-ginsengs, 121 DEG C of autoclave sterilization 40min in culture medium are converted to 50ml；
(4) microbe conversion：The slant strains G11-7 grown in step 1 is taken, strain surface spore is scraped down and is made one
Determine the spore suspension of concentration, adding 10ml spore suspensions into the former powder of pseudo-ginseng by pretreatment carries out bioconversion, turns
It is 30 DEG C to change temperature, and rotating speed is 120r/min, and transformation time is 48h；
(5) extraction of ginsenoside：Mycelium and the former powder of pseudo-ginseng are removed by centrifuging, supernatant is obtained, by positive fourth
Alcohol carries out equal-volume extraction with supernatant, repeats three times, merges organic phase and is concentrated under reduced pressure, obtains ginsenoside and slightly carry
Thing.
Embodiment 4：The raw sickle-like bacteria in middle level (Fusarium proliferatum) G11-7 bacterial strains orientation conversion three of the present invention
Seven ginsenoside Rb1s are Rd detection.
High performance liquid chromatography detection converted product step is as follows:By ginsenoside crude extract after chromatogram methanol dilution, use
High performance liquid chromatograph carries out analysis detection.Chromatographic condition be the chromatographs of Agilent 1100, HiQ sil C18W (5 μm) chromatographic column, mobile phase：Water (A)-acetonitrile (B), 30 DEG C of column temperature；Detection wavelength 203nm, flow velocity
0.8mL/min, the μ L of sample size 10, condition of gradient elution：0-5min, 25-30% (B)；5-15min, 30-40% (B)；15-
16min, 40-41% (B)；16-20min, 41-42% (B)；20-30min, 42-50% (B)；30-35min, 50-60% (B)；
35-60min, 60-40% (B)
Testing result shows：Fermentative bioconversion, every liter of fermentation are carried out using pigeonpea endogenetic fungus G11-7 bacterial strains of the present invention
The content of ginsenoside Rd increases to 478.44mg [as shown in Figure 6] from 118.36mg in liquid.Meanwhile, convert panax ginsenoside
Short, high conversion rate, green non-pollution the time required to Rb1 is Rd, can be achieved large-scale production, with higher application value.
Claims (4)
1. pigeonpea endogenetic fungus and its application of one plant of Efficient Conversion panax ginsenoside Rb1 for Rd, it is characterised in that：The layer
Raw sickle-like bacteria (Fusarium proliferatum) G11-7, is Fungi Imperfecti (Imperfecti fungi), Moniliales
(Moniliales), Tuberculariaceae (Tuberculariaceae), Fusarium (Fusarium).
2. pigeonpea endogenetic fungus and its application of the one plant of Efficient Conversion ginsenoside Rb1 according to claim 1 for Rd, its
Endogenetic fungus raw sickle-like bacteria (Fusarium proliferatum) G11-7 of layer is characterised by, in Chinese microorganism strain preservation
Administration committee's common micro-organisms center preservation, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number
For CGMCC No:13195, preservation date is on December 7th, 2016.
3. pigeonpea endogenetic fungus and its answer that one plant of Efficient Conversion panax ginsenoside Rb1 according to claim 1 is Rd
With, it is characterised in that the endogenetic fungus is when PDA solid cultures are based on 28 DEG C of cultures, the white purplish of bacterium colony, mitogenetic spore
Son is oval or bar-shaped monospore, scattered or with pionnotes consor.
4. pigeonpea endogenetic fungus G11-7 according to claim 1, it, which is metabolized the cellulase produced, has people in pseudo-ginseng
Ginseng saponin(e Rb1 is biologically converted into the activity of ginsenoside Rd.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019119992A1 (en) * | 2017-12-21 | 2019-06-27 | 德州学院 | Endophytic fungus of ginkgo biloba and metabolite product and application thereof |
| CN113717860A (en) * | 2021-07-07 | 2021-11-30 | 昆明理工大学 | Application of Talaromyces flavidus in conversion of panax notoginseng saponins into low-polarity ginsenoside |
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| CN101481725A (en) * | 2009-01-21 | 2009-07-15 | 华东理工大学 | Method for preparing ginseng saponin F2 by enzymatic hydrolysis of ginseng saponin Rb1 |
| CN103255193A (en) * | 2013-03-05 | 2013-08-21 | 吉林农业大学 | Ginsenoside conversion method by use of ginseng endophytic Paenibacillus polymyxa |
| CN105838613A (en) * | 2015-01-14 | 2016-08-10 | 东北林业大学 | Pigeon pea endophytic fungi high in yield of flavipin and application of pigeon pea endophytic fungi |
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2017
- 2017-03-01 CN CN201710115419.5A patent/CN107312720B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101481725A (en) * | 2009-01-21 | 2009-07-15 | 华东理工大学 | Method for preparing ginseng saponin F2 by enzymatic hydrolysis of ginseng saponin Rb1 |
| CN103255193A (en) * | 2013-03-05 | 2013-08-21 | 吉林农业大学 | Ginsenoside conversion method by use of ginseng endophytic Paenibacillus polymyxa |
| CN105838613A (en) * | 2015-01-14 | 2016-08-10 | 东北林业大学 | Pigeon pea endophytic fungi high in yield of flavipin and application of pigeon pea endophytic fungi |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019119992A1 (en) * | 2017-12-21 | 2019-06-27 | 德州学院 | Endophytic fungus of ginkgo biloba and metabolite product and application thereof |
| US11332707B2 (en) | 2017-12-21 | 2022-05-17 | Dezhou University | Endophytic fungus from gingko, metabolite product and use thereof |
| CN113717860A (en) * | 2021-07-07 | 2021-11-30 | 昆明理工大学 | Application of Talaromyces flavidus in conversion of panax notoginseng saponins into low-polarity ginsenoside |
| CN113717860B (en) * | 2021-07-07 | 2023-05-16 | 昆明理工大学 | Application of Huang Lanzhuang bacteria in conversion of total saponins of Notoginseng radix into small-polarity ginsenoside |
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