CN104651238B - One plant of Cercospora endogenetic fungal bacterial strain and its application - Google Patents
One plant of Cercospora endogenetic fungal bacterial strain and its application Download PDFInfo
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Abstract
The invention discloses one plant of Cercospora endogenetic fungal bacterial strain and its application.The fungi of the generation secondary metabolite is Cercospora (Cercospora sp.nov.) WDL2, is CCTCC NO in the deposit number of China typical culture collection center：M 2014518.Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO that the present invention is provided：The mycelial growth speed of M 2014518 is 0.22g/d, the content of polysaccharide, flavones, total triterpene and total phenol is respectively 74.452mg/g, 1.250mg/g, 4.395mg/g and 3.343mg/g in its mycelium (dry weight), by Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO：The contents of flavones, total triterpene and total phenol in 10 days its zymotic fluids of fermented and cultured under most suitable conditions of flask fermentation of M 2014518 are respectively 0.668mg/L, 0.633mg/L and 2.124mg/L.Show that the bacterial strain can produce polysaccharide, flavones, total triterpene and total phenol, there is important application in terms of active pharmaceutical ingredient is produced.
Description
Technical field
The present invention relates to one plant of Cercospora endogenetic fungal bacterial strain and its application, the secondary metabolite in microorganism field
Can be four kinds, three kinds, two kinds or a kind of in polysaccharide, flavones, triterpene and total phenol.
Background technology
The metabolite of plant can be divided into two classes, that is, come into being metabolite and secondary metabolite.Nascent metabolite is
Refer to and maintain matter and energy metabolism necessary to plant normal activities, such as lipid, amino acid, nucleic acid and theirs is more
Aggressiveness etc., the molecular weight of these materials is general very big, is also called macromolecular compound.Secondary metabolite is produced by secondary metabolism
The nonessential small molecular organic compounds that a raw class cell activities or growth and development of plants normally run (traditionally claim
Be natural products), its produce and distribution generally have kind, organ, organize and period of growing specificity, secondary metabolism
Product can be divided into Phenylpropanoid Glycosides class, quinones, flavonoids, tanninses, terpenoid, steroidal and its glucoside, the major class of alkaloid seven.Plant certain
In individual developmental stage or certain organ, secondary metabolite is likely to become the main component of metabolic pool, such as rubber tree produces big
The content of Stevioside is up to more than the 10% of dry weight in amount rubber and stevia rebaudian leaf.
Direct or indirect extraction is the Main Means that current people obtain natural products in plant, but with the increasing of population
The increase of many market demands, directly extracts secondary metabolite from plant and has been difficult to meet demand, so development is new
Efficiently produce these important secondary metabolite methods extremely urgent.Endogenetic fungus are lives of surviving and complete in host
The fungi in cycle, the endogenetic fungus colonized in for a long time on certain plant generally can be with the host plant coevolution, host plant
The phenomenon of genetic recombination is there may be between its endogenetic fungus, so that the endogenetic fungus on host plant can be produced and place
Main plant identical metabolite.Have 51% in the natural products of discovered in recent years from plant endogenesis epiphyte, 38% from soil
Earth microorganism, 11% come from plant and animal, it is clear that endogenetic fungus oneself turn into natural products valuable source.
Wild Peas living environment is original, complexity, and population diversity is high, and the richness and diversity of its endogenetic fungus are compared with it
Its species is more superior.Some more original and secondary metabolites can obtain by the separation to wild Peas endogenetic fungus many
The endogenetic fungus of sample.Cercospora (Cercospora) fungi can cause lawn leaf spot, common are 4 types：Cause Jian's stock
Creeping bentgrass tail spore bacterium (C.agrostidis) of clever leaf spot, fescue grass tail spore bacterium (C.festucae) for causing fescue grass leaf spot,
The C.seminalis for the causing buffalograss and Bermuda grass leaf spot and C.fusimaculans. for causing Herba Stenotaphri helferi leaf spot.Also have
On tail spore bacterium as water hyacinth biocontrol microorganisms application, but on Cercospora endogenetic fungus secondary metabolite analysis report very
It is few.
The content of the invention
How the technical problems to be solved by the invention are using micro-organisms secondary metabolite, the secondary metabolism
Product is four kinds, three kinds, two kinds or a kind of in polysaccharide, flavones, total triterpene and total phenol.
In order to solve the above technical problems, the invention provides the endogenetic fungus that a plant can produce secondary metabolite, institute
It is four kinds, three kinds, two kinds in polysaccharide, flavones, total triterpene and total phenol or a kind of to state secondary metabolite.
The endogenetic fungus that the present invention is provided are Cercospora (Cercospora sp.nov.) WDL2, and the bacterial strain is in 2014
October 27 was preserved in China typical culture collection center, and (abbreviation CCTCC, address is：Wuhan, China Wuhan University), preservation
Numbering is CCTCC NO：M 2014518.
It is a further object to provide a kind of microbial inoculum, the active component of the microbial inoculum is described Cercospora WDL2
(Cercospora sp.nov.WDL2)CCTCC NO：M 2014518.
Above-mentioned microbial inoculum can be to produce four kinds, three kinds, two kinds in secondary metabolite polysaccharide, flavones, total triterpene and total phenol
Or a kind of microbial inoculum.
The microbial inoculum can also include carrier.The carrier can be solid carrier or liquid-carrier.The solid carrier can
It is mineral material, vegetable material or macromolecular compound；The mineral material can be clay, talcum, kaolin, montmorillonite, white
At least one in carbon, zeolite, silica and diatomite；The vegetable material can be at least in corn flour, bean powder and starch
Kind；The macromolecular compound can be polyvinyl alcohol and/or polyglycols.The liquid-carrier can be organic solvent, vegetable oil, ore deposit
Thing oil or water；The organic solvent can be decane and/or dodecane.In the microbial inoculum, the active component can be being cultured
Living cells, the zymotic fluid of living cells, the form of the mixture of the filtrate of cell culture or cell and filtrate be present.Described group
The formulation of compound can be various formulations, such as liquor, emulsion, suspending agent, pulvis, granule, wettable powder or water dispersible granules.
As needed, surfactant (such as polysorbas20, Tween 80), adhesive, stabilization can be also added in the microbial inoculum
Agent (such as antioxidant), pH adjusting agent.
Cercospora WDL2 (Cercospora sp.nov.WDL2) the CCTCC NO：M 2014518 is producing secondary generation
Thank four kinds in product polysaccharide, flavones, total triterpene and total phenol, three kinds, two kinds or a kind of application falls within protection of the invention
Scope.
It is described with Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO：M 2014518 is active component
Four kind, three kind, two kind or a kind of application of the microbial inoculum in production secondary metabolite polysaccharide, flavones, total triterpene and total phenol
Fall within protection scope of the present invention.
It is also another object of the present invention to provide one kind culture Cercospora WDL2 (Cercospora sp.nov.WDL2)
CCTCC NO：The method of M 2014518.
Culture Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO provided by the present invention：M
2014518 method, including by Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO：M 2014518 with
The step of being cultivated in the culture medium for cultivating tail spore bacterium.
A further object of the present invention is to provide one kind with Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC
NO：M 2014518 is the preparation method of the microbial inoculum of active component.
The preparation method of above-mentioned microbial inoculum provided by the present invention, comprises the following steps：By described Cercospora WDL2
(Cercospora sp.nov.WDL2)CCTCC NO：M 2014518 obtains the microbial inoculum as active component.
To Heilongjiang Province of China Acheng District forest farm gather wild pea blade endogenetic fungus separation, be likely to be obtained some compared with
It is original and secondary metabolite endogenetic fungus vdiverse in function.Separation screening of the present invention obtains producing secondary metabolite
Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO：M 2014518.
It is demonstrated experimentally that Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO：The mycelium of M 2014518
(dry weight) growth rate is 0.22g/d, and the content of polysaccharide, flavones, total triterpene and total phenol is respectively in its mycelium (dry weight)
74.452mg/g, 1.250mg/g, 4.395mg/g and 3.343mg/g, under most suitable conditions of flask fermentation fermented and cultured 10 days its
The content of flavones, total triterpene and total phenol is respectively 0.668mg/L, 0.633mg/L and 2.124mg/L in zymotic fluid.This shows this
Bacterial strain can produce polysaccharide, flavones, total triterpene and total phenol, have important application in terms of secondary metabolite is produced.
Preservation explanation
Strain name：Cercospora
Latin name：Cercospora sp.nov.
Strain number：WDL2
Preservation mechanism：China typical culture collection center
Preservation mechanism is referred to as：CCTCC
Address：Wuhan, China Wuhan University
Preservation date：On October 27th, 2014
Collection is registered on the books numbering：CCTCC NO：M 2014518
Brief description of the drawings
Fig. 1 is Cercospora (Cercospora sp.nov.) mycelia of WDL2 under 10 times of light microscopes of growth 4 days
Form.
Fig. 2 is Cercospora (Cercospora sp.nov.) mycelia of WDL2 under 40 times of light microscopes of growth 7 days
Form.
Fig. 3 is the colonial morphology of Cercospora (Cercospora sp.nov.) WDL2 for growing 15 days.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Embodiment 1, Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO：The separation of M 2014518, mirror
Fixed and preservation
1st, the separation of endogenetic fungus WDL2
Wild pea blade (picking up from Heilongjiang Province of China Acheng District Ya Gou forest farms) is carried out disinfection.Chloramphenicol is added
2% malt flour agar medium makes its content for 50mg/L, obtains malt flour chloramphenicol flat board；It is made of PDA solid mediums
PDA solid plates.Wild pea blade after above-mentioned sterilization is cut into 0.5cm2Fritter be placed on malt flour chloramphenicol flat board,
25 DEG C of dark culturings 5 days.The Endophytic Fungal Hyphae grown from wild pea leaf tissue notching edge is used into oese one by one
Being connected to carries out purifying agaric on PDA solid plates.The wherein one plant endogenetic fungus that will be screened are named as endogenetic fungus WDL2.
2nd, the identification of endogenetic fungus WDL2
The endogenetic fungus WDL2 for obtaining is separated and purified from the following aspects authentication step one：
2.1st, Morphological Identification
The endogenetic fungus WDL2 for obtaining will be separated and purify in exponential phase and bacterium colony size stabilization, above-mentioned steps one
Carry out single bacterium colony state observation, the main size including bacterium colony, color, transparency, bacterium colony surface state (whether flat, projection,
Fold, depression etc.), colony edge feature (whether neat, irregular, radial etc.), produce spore time etc..Bacterium colony mode of appearance is special
Collection is levied using Canon 60D cameras (18-200mm IS, Japan), technical parameter is：APS-C specifications are digital single anti-；1800 is universal
Effect pixel；10 Zoom Lens.
To the endogenetic fungus WDL2 in exponential phase, observation by light microscope mycelia is used after smear staining
Form, the microstructure (such as stroma, conidiophore, conidium) of surface state, mycelia including mycelia.Bacterium colony shows
Microcosmic examining uses upright biomicroscope Axioskop2plusFL (Zeiss, Germany), technical parameter：Achromatism-aplanasia is more
Function condenser；Illuminator is 100W Halogen lamp LEDs, Kohler illumination；Five grades of fluorescence spectrophotometer devices；HB050, HB0103 light source.Using
Axioplan 2imaging MOT softwares carry out IMAQ with analysis.
Result shows (Fig. 1, Fig. 2 and Fig. 3) that above-mentioned steps one separate and purify the endogenetic fungus WDL2 for obtaining with as follows
Morphological feature：Bacterium colony projection, front white, back side dark brown, surface is smooth, and there are fold, culture medium yellowing, bacterium colony in edge
Thick 2-3mm, growth is more slow during with PDA solid medium cultures, and 16 days colony diameters of culture are 4 centimetres.Microstructure is seen
Display is examined, has larger stroma, stroma is subsphaeroidal, 80 μm~160 μm of diameter.Conidiophore list is given birth to or many intensive fasciations are in son
Light yellow on seat, color and luster is uneven, and conidiophore is (10 μm~560 μm) more long, every many, the clear spacing of barrier film it is short (5 μm~
40 1.5 μm of μ ms~2.5 μm), 1.4 μm~2.4 μm of width rule has less branch, more uprightly, top flat round.Conidium
Cylindrical type is linear, and upright or bending is cyclic (15 18 μm of μ m), and top smooths, more unobvious barrier film (8 μm of -22 μ m 0.8-
2.2 μm), fawn, base portion turbination is truncate.Long and narrow (30 μm~480um × 0.8 μm~2.2 μm).Spore trace scar is obvious
Thicken, it is wide 1.2 μm~2.5 μm.
With separate Cercospora fungi and be distinguished as at present, have a larger stroma, a diameter of 80 μm~160 μm, conidiophore compared with
(10 μm~560 μm) long, width is relatively regular (1.4 μm~2.4 μm), every many, short (5 μm~40 1.5 μm of the μ ms of the clear spacing of barrier film
~2.5 μm), conidium is cylindrical or linear, and upright or bending is cyclic (15 18 μm of μ m), more unobvious barrier film (8 μm~22
0.8 μm~2.2 μm of μ m), fawn, long and narrow (30 μm~480 0.8 μm of μ ms~2.2 μm).Spore trace scar substantially thickeies,
It is wide 1.2 μm~2.5 μm.
2.2nd, molecular biology identification
Endogenetic fungus WDL2 genomic DNAs are extracted with CTAB methods.Made with the endogenetic fungus WDL2 genomic DNAs of said extracted
It is amplification template, with fungi ribosomes rDNA areas universal primers ITS1：5 '-CTTGGTCATTTAGACGAAGTAA-3 ' and ITS4：
5 '-GCATATCAATAAGCGGAGGA-3 ' enter performing PCR reaction for primer.Response procedures are：94 DEG C of denaturation 40s, 55 DEG C of annealing
50s, 72 DEG C of extension 1min, totally 35 circulations.PCR primer to obtaining is sequenced, and sequencing result shows endogenetic fungus WDL2
RDNA-ITS sequences as shown in SEQ ID No.1, by published rDNA- in the sequence and NCBI and EzTaxon databases
ITS sequence carries out online sequence analysis, as a result shows endogenetic fungus WDL2 and Cercospora (Cercospora lagenariae
Strain) fungal nucleic acid sequence homology highest, similitude is 95%.
According to above-mentioned form, molecular biological analysis and rDNA-ITS sequence homology analysis results, step one is separated
The endogenetic fungus WDL2 that purifying is obtained is accredited as the novel species of Cercospora, and it is Cercospora (Cercospora sp.nov.) to name.Should
Endogenetic fungus WDL2 has been preserved in China typical culture collection center on October 27th, 2014, and (abbreviation CCTCC, address is：
Wuhan, China Wuhan University), deposit number is CCTCC NO：M 2014518.The full name of endogenetic fungus WDL2 is Cercospora WDL2
(Cercospora sp.nov.WDL2)CCTCC NO：M 2014518, referred to as Cercospora (Cercospora sp.nov.)
WDL2。
3rd, the culture presevation of Cercospora (Cercospora sp.nov.) WDL2
By liquid storage medium sterilizing (liquid storage medium：Solute and its concentration are：15% (percent by volume is dense
Degree) glycerine, glucose 10g/L, yeast extract 1g/L, casein hydrolysate 1g/L；Solvent is deionized water；PH natures),
1mL liquid storage mediums, picking Cercospora (Cercospora sp.nov.) WDL2 bacterium are added in aseptic 1.5mL centrifuge tubes
Fall in edge mycelium inoculation to preservative fluid, cover centrifugation lid and sealed with sealed membrane, after being put into 4 DEG C of refrigerator precoolings, place into-
Preservation in 80 DEG C of refrigerators.
The research of embodiment 2, Cercospora (Cercospora sp.nov.) most suitable conditions of flask fermentation of WDL2 and mycelium
Growth rate measurment
Using mycelial biomass (dry weight) as measurement index, i.e., with the time as abscissa, with dry mycelial weight as ordinate,
Growth curve is drawn, Cercospora (Cercospora sp.nov.) most suitable conditions of flask fermentation of WDL2 bacterial strains is studied.Knot
Fruit shows that the suitable fermentation condition of Cercospora (Cercospora sp.nov.) WDL2 is：Using PDA liquid medium in triangle
Shake flask fermentation culture is carried out in bottle, the cultivation temperature of fermented and cultured can be 20 DEG C -30 DEG C.Using 250mL triangular flasks as fermentation
Container, the liquid amount of PDA liquid medium can be 100mL-150mL in 250mL triangular flasks, carry out rotation concussion and cultivate, shaking table
Rotating speed can be 120rpm-180rpm, radius of turn is 13mm, the incubation time of fermented and cultured concretely -15 days 8 days.
The most suitable conditions of flask fermentation of Cercospora (Cercospora sp.nov.) WDL2 is：Using PDA liquid medium
Shake flask fermentation culture, 25 DEG C of the cultivation temperature of fermented and cultured are carried out in triangular flask.Held as fermentation using 250mL triangular flasks
Device, the liquid amount of PDA liquid medium is 120mL in 250mL triangular flasks, carries out rotation concussion and cultivate, the rotating speed of shaking table
160rpm, radius of turn is 13mm, and the incubation time of fermented and cultured is 10 days.
Result shows：The fermented and cultured Cercospora (Cercospora sp.nov.) under above-mentioned most suitable conditions of flask fermentation
WDL2, is centrifuged zymotic fluid after 10 days, collect mycelium, and the mycelium that will be obtained is named as mycelium 1；Mycelium 1 is placed in tin
On foil paper in an oven 60 DEG C dry the mycelium that is obtained to constant weight and be named as mycelium 2.Mycelium 2 is put into cooling in drier
Weigh again, as mycelial biomass (dry weight).
Result shows that the biomass (dry weight) that assay balance weighs mycelium 2 is 2.2g, can be calculated mycelial growth speed
Rate is 0.22g/d.
Secondary metabolite contains in embodiment 3, Cercospora (Cercospora sp.nov.) WDL2 mycelium and zymotic fluid
It is fixed to measure
In following embodiments Cercospora used (Cercospora sp.nov.) WDL2 zymotic fluids be in embodiment 2 most
Fermented and cultured Cercospora WDL2 (Cercospora sp.nov.WDL2) CCTCC NO under suitable conditions of flask fermentation：M201451810
My god, the thalline during filtering fermentation liquor removes zymotic fluid is collected, collect remaining liquid abbreviation zymotic fluid 1；Zymotic fluid 1 is rotated
Evaporimeter concentrates 5 times and obtains concentrated broth, and the concentrated broth is named as into zymotic fluid 2.
1st, measurement of the polysaccharide content in Cercospora (Cercospora sp.nov.) WDL2 mycelium
1.1st, the drafting of standard curve：The accurate DEXTROSE ANHYDROUS reference substance (Beijing Century for weighing 105 DEG C of dryings to constant weight
AudioCodes Bioisystech Co., Ltd) 0.500g, add deionized water to be configured to 500 μ g/ml glucose standards solutions.It is accurate respectively
Draw 50 μ l, 150 μ l, 250 μ l, 350 μ l, 450 μ l standard liquids to be placed in the graded tube of 10ml, complemented to deionized water
2.5mL, then (solute and its concentration are to be separately added into 6.5ml anthrones-concentrated sulfuric acid solution：Anthrone 2.0g/L；Solvent：The concentrated sulfuric acid；
pH：It is natural), after boiling water bath 10min, cool down 10min.
Determine absorbance of each concentration standard liquid at 620nm, 3 repetitions respectively with spectrophotometer.With grape
Sugared content (μ g/mL) is abscissa, OD620It is ordinate to be worth, and draws glucose standard curve.Gained glucose assays standard is bent
Line is x=260.08y-4.5448 (R2=0.996), wherein y is OD620Value, x is glucose content (μ g/mL), and glucose exists
25-225 μ g/mL linear relationships are good.
1.2nd, the measure of Cercospora (Cercospora sp.nov.) WDL mycelium polysaccharides contents：By the bacterium in embodiment 2
Filament 2 is clayed into power, and is accurately weighed 0.200g and is added 5ml deionized waters to boil 30min, and supernatant is poured into triangular flask and collected by centrifugation,
5ml deionized waters are continuously added in precipitation and boils 30min, filtered, filtrate is poured into same triangular flask and is collected, and uses deionized water
It is settled to 25ml and obtains final product Cercospora (Cercospora sp.nov.) WDL2 mycelium polysaccharides extract solutions, abbreviation polysaccharide extraction liquid 1.
0.2ml polysaccharide extraction liquids 1 are taken, 2.5mL is complemented to deionized water, 6.5ml anthrones-concentrated sulfuric acid solution is mixed, boiling water bath 10min
Afterwards, 10min is cooled down.The absorbance at 620nm is determined under spectrophotometer, is calculated according to glucose assays standard curve
The content of polysaccharide in polysaccharide extraction liquid 1, further calculates polysaccharide in Cercospora (Cercospora sp.nov.) WDL2 mycelium
Content.
Result shows that the absorbance that polysaccharide extraction liquid 1 is determined at 620nm is 0.4755, according to glucose assays mark
The content that directrix curve calculates polysaccharide in polysaccharide extraction liquid 1 is 119.123 μ g/mL, i.e. Cercospora (Cercospora sp.nov.)
The content of polysaccharide is 74.452mg/g in WDL2 mycelium.
2nd, in Cercospora (Cercospora sp.nov.) WDL2 mycelium and zymotic fluid flavones content measure
2.1st, the drafting of standard curve：Accurately weigh control substance of Rutin (Beijing Century AudioCodes Bioisystech Co., Ltd)
10.0mg adds the 50% ethanol water dissolving of 4ml in 10mL volumetric flasks, is then settled to 50% ethanol water
10mL, is made into 1000mg/L rutin mother liquors.It is accurate draw the μ l of rutin mother liquor 0,250 μ l, 400 μ l, 500 μ l, 600 μ l, 750 μ l,
1000 μ l are respectively placed in 25ml volumetric flasks, be sequentially added into 1mL5% sodium nitrite solutions, 1mL10% aluminum nitrate solutions and
10mL10% NaOH, 50% ethanol water is settled to 25ml, shakes up standing 15min.
Determine absorbance of each concentration standard liquid at 550nm, 3 repetitions respectively with spectrophotometer.With rutin
Content (μ g/mL) is abscissa, OD550It is ordinate to be worth, and draws rutin standard curve.Gained rutin bioassay standard curve is x=
0.0875y+0.0002(R2=0.9997), wherein y is OD550Value, x is rutin content (μ g/mL), and rutin is in 0-1.0mg/mL
Linear relationship is good.
2.2nd, the measure of Cercospora (Cercospora sp.nov.) WDL2 mycelium flavones contents：By in embodiment 2
Mycelium 2 is clayed into power, and is accurately weighed 0.200g and is placed in the triangular flask of 50ml, adds the ethanol waters of 10ml 50%, will be held
Measuring bottle vibrates 90min in being placed on ultrasonic wave bath, and filtering, filtrate is settled to 25ml and obtains final product Cercospora with 50% ethanol water
(Cercospora sp.nov.) WDL2 mycelium flavone extractives, are named as flavone extractive 1.4ml flavone extractives 1 are taken,
It is sequentially added into the sodium nitrite solutions of 1mL 5%, the aluminum nitrate solutions of 1mL 10% and the NaOH of 10mL 10%, 50% second
Alcohol solution is settled to 25ml, shakes up standing 15min.The absorbance at 550nm, 3 weights are determined under spectrophotometer
It is multiple.The content of flavones in flavone extractive 1 is calculated according to rutin bioassay standard curve.
Result shows that the absorbance that polysaccharide extraction liquid 1 is determined at 550nm is 0.11, bent according to rutin bioassay standard
The content of flavones is 0.010mg/mL, i.e. Cercospora (Cercospora sp.nov.) WDL2 bacterium in line computation flavone extractive 1
The content of flavones is 1.250mg/g in filament.
2.3rd, in Cercospora (Cercospora sp.nov.) WDL2 zymotic fluids (i.e. zymotic fluid 1) flavones content measure：
Take the absolute ethyl alcohol of 5ml zymotic fluids 2 and be settled to 10ml and obtain final product Cercospora (Cercospora sp.nov.) WDL2 zymotic fluid flavones
Extract solution, is named as flavone extractive 2.4ml flavone extractives 2 are taken, the sodium nitrite solutions of 1mL 5%, 1mL is sequentially added into
10% aluminum nitrate solution and 10mL10% NaOH, 50% ethanol water are settled to 25ml, shake up standing 15min.Dividing
The absorbance at 550nm is determined under light photometer, 3 repetitions, OD values 0.074 are calculated according to rutin bioassay standard curve
The content of flavones in flavone extractive 2, the content for further calculating flavones in zymotic fluid 1 is 0.668mg/L.
3rd, in Cercospora (Cercospora sp.nov.) WDL2 mycelium and zymotic fluid total triterpene contentses measure
3.1st, accurate oleanolic acid reference substance (the Beijing Century AudioCodes Bioisystech Co., Ltd) 2.5mg that weighs holds in 25ml
In measuring bottle, 95% ethanol water dissolves and is settled to 25ml, is made into 0.1mg/mL oleanolic acid mother liquors.It is accurate to draw olive
Acid mother liquor 0.025ml, 0.1ml, 0.2ml, 0.5ml, 1.0ml, 2.5ml are respectively placed in 5ml volumetric flasks, and 95% ethanol is water-soluble
Liquid is settled to 5ml.Take 100 μ l respectively to be placed in 10ml centrifuge tubes, 70 DEG C of water bath methods.To adding 200 μ l in above-mentioned centrifuge tube
5% vanillic aldehyde-glacial acetic acid (solute and its concentration are：Vanillic aldehyde 50.0g/L；Solvent：Glacial acetic acid；pH：It is natural) and 800 μ l it is high
Chloric acid (Tianjin Ward Hua Yan scientific ＆ technical corporation product), 70 DEG C of water-bath 15min are put into rapidly after cooling down in ice and use ethyl acetate constant volume
To 5ml.Determine absorbance of each concentration standard liquid at 551nm, 3 repetitions respectively with spectrophotometer.With olive
Acid content (mg/mL) is abscissa, OD551It is ordinate to be worth, and draws oleanolic acid standard curve.Gained oleanolic acid determines mark
Directrix curve is x=43.044y-0.6438 (R2=0.9970), wherein y is OD551Value, x is content of oleanolic acid (mg/mL), together
Pier tartaric acid content is good in 0.5-50mg/L linear relationships.
3.2nd, the measure of Cercospora (Cercospora sp.nov.) WDL2 mycelium total triterpene contentses：By in embodiment 2
Mycelium 2 clay into power, accurately weigh 0.200g add the ethanol waters of 2ml 95%, 70 DEG C of waters bath with thermostatic control 1h, Ran Hou
40min is vibrated in ultrasonic wave, 100 μ l is taken and is placed in 10ml centrifuge tubes, 70 DEG C of water bath methods.To adding 200 μ in above-mentioned centrifuge tube
(solute and its concentration are 5% vanillic aldehyde of l-glacial acetic acid：Vanillic aldehyde 50.0g/L；Solvent：Glacial acetic acid；pH：It is natural) and 800 μ l
Perchloric acid (Tianjin Ward Hua Yan scientific ＆ technical corporation product), 70 DEG C of water-bath 15min, it is fixed with ethyl acetate after cooling down in ice to be put into rapidly
Hold 5ml and obtain final product Cercospora (Cercospora sp.nov.) WDL2 mycelium total triterpene extract solutions, be named as total triterpene extraction
Liquid 1.The absorbance at 551nm, 3 repetitions are determined under spectrophotometer.According to oleanolic acid bioassay standard curve meter
The content of total triterpene in total triterpene extract solution 1 is calculated, Cercospora (Cercospora sp.nov.) WDL2 mycelium are further calculated
The content of middle total triterpene.
Result shows that the absorbance that total triterpene extract solution 1 is determined at 551nm is 1.036, according to bioassay standard curve
The content for calculating total triterpene in total triterpene extract solution 1 is 43.950 μ g/mL, further calculates Cercospora (Cercospora
Sp.nov.) content of total triterpene is 4.395mg/g in WDL2 mycelium.
3.3rd, in Cercospora (Cercospora sp.nov.) WDL2 zymotic fluids (i.e. zymotic fluid 1) total triterpene contentses survey
It is fixed：4ml zymotic fluids 2 are taken, water bath method adds the dissolving of 2ml95% ethanol waters, therefrom takes 100 μ l mixed liquors and be placed in 10ml
In centrifuge tube, to 5% vanillic aldehyde-glacial acetic acid that 200 μ l are added in above-mentioned centrifuge tube and 800 μ l perchloric acid, 70 DEG C of water-baths
15min, is put into rapidly after cooling down in ice and obtains final product Cercospora (Cercospora sp.nov.) WDL2 with ethyl acetate constant volume to 5ml
Zymotic fluid total triterpene extract solution, is named as total triterpene extract solution 2.The absorbance at 551nm is determined under spectrophotometer,
3 repetitions.The content of total triterpene in total triterpene extract solution 2 is calculated according to oleanolic acid bioassay standard curve, hair is further calculated
The content of total triterpene in zymotic fluid 1.
Result shows that the absorbance that total triterpene extract solution 2 is determined at 551nm is 0.162, according to bioassay standard curve
The content for calculating total triterpene in total triterpene extract solution 2 is 6.329 μ g/mL, and the content for further calculating total triterpene in zymotic fluid 1 is
0.633mg/L。
4th, in Cercospora (Cercospora sp.nov.) WDL2 mycelium and zymotic fluid total phenol content measure
4.1st, the drafting of standard curve：Accurately weigh gallic acid reference substance (the limited public affairs of Beijing Century AudioCodes biotechnology
Department's product) 25.0mg in 25ml volumetric flasks, 25ml is settled to deionized water, is made into 1mg/ml mother solution of gallic acid.Accurately
Mother solution of gallic acid 0,0.05,0.1,0.25,0.5,1.0ml is drawn to be respectively placed in the volumetric flask of 25mL, plus deionized water is extremely
Volume is 5.0mI, adds the ferrous tartrate solution (composition of ferrous tartrate solution：Solute and its concentration are：1g/L sulfuric acid
Ferrous, 5g/L sodium potassium tartrate tetrahydrates；Solvent：Deionized water；PH natures) 5.0ml, it is settled to the PBS of pH7.5
25ml, mixes and stands 15min.
Determine absorbance of each concentration standard liquid at 540nm, 3 repetitions respectively with spectrophotometer.Not eat
The concentration of sub- acid solution is abscissa, OD540It is ordinate to be worth, and draws gallic acid standard curve.Gained gallic acid determines mark
Directrix curve is x=0.6029y+0.0072 (R2=0.997), wherein y is OD540Value, x is gallic acid solution concentration (mg/
ML), gallic acid concentration linear relationship in 0-0.7mg/mL is good.
4.2nd, the measure of Cercospora (Cercospora sp.nov.) WDL2 mycelium total phenol contents：By in embodiment 2
Mycelium 2 is clayed into power, and accurately weighs 0.200g in 50ml triangular flasks, adds the ethanol waters of 7ml 60% to be placed on ultrasonic wave
45min is extracted, supernatant is transferred to new 50ml triangular flasks, remaining solid powder in triangular flask is so repeated 3 times.Will be upper
State the supernatant for obtaining for 3 times and be placed in 70 DEG C of water bath methods in same triangular flask, being settled to 15ml with deionized water obtains final product tail
Spore belongs to the total phenol extraction liquid of (Cercospora sp.nov.) WDL2 mycelium, is named as total phenol extraction liquid 1.Take the total phenol extractions of 5ml
Liquid 1 plus 4ml deionized waters and 5ml ferrous tartrate solution, 25mI is settled to the PBS of pH7.5.In light splitting
The absorbance at 540nm, 3 repetitions are determined under photometer.Total phenol extraction is calculated according to gallic acid bioassay standard curve
The content of total phenol, further calculates the content of the total phenol of total phenol extraction liquid 1 in liquid 1.
Result shows that the absorbance that total phenol extraction liquid 1 is determined at 540nm is 0.062, according to bioassay standard curve meter
Calculate in total phenol extraction liquid 1 content of total phenol be 445.8 μ g/mL, further calculate Cercospora (Cercospora sp.nov.)
The content of total phenol is 3.343mg/g in WDL2 mycelium.
4.3rd, in Cercospora (Cercospora sp.nov.) WDL2 zymotic fluids total phenol content measure：Take 5ml zymotic fluids 2
Plus 4ml deionized waters and 5ml ferrous tartrate solution, it is settled to 25mI with the PBS of pH7.5.In spectrophotometric
The lower absorbance determined at 540nm of meter, 3 repetitions.According in the gallic acid bioassay standard curve total phenol extraction liquid 2 of calculating
The content of total phenol, further calculates the content of total phenol in zymotic fluid 1.
Result shows that the absorbance that total phenol extraction liquid 2 is determined at 540nm is 0.076, according to bioassay standard curve meter
The content for calculating total phenol in total phenol extraction liquid 2 is 53.17 μ g/mL, and the content for further calculating total phenol in zymotic fluid 1 is 2.124mg/
L。
Claims (6)
1. Cercospora（Cercospora sp.nov.）WDL2 bacterial strains, its deposit number in China typical culture collection center
It is CCTCC NO：M 2014518.
2. a kind of microbial inoculum, it is characterised in that：The active component of the microbial inoculum is the Cercospora WDL2 described in claim 1
（Cercospora sp.nov. WDL2）CCTCC NO：The bacterial strains of M 2014518.
3. the Cercospora WDL2 described in claim 1（Cercospora sp.nov. WDL2）CCTCC NO：The bacterium of M 2014518
Application of the strain in secondary metabolite is produced；The secondary metabolite is four in polysaccharide, flavones, total triterpene and total phenol
Kind, three kinds, two kinds or one kind.
4. application of the microbial inoculum described in claim 2 in secondary metabolite is produced；The secondary metabolite is polysaccharide, Huang
Four kinds, three kinds, two kinds or a kind of in ketone, total triterpene and total phenol.
5. Cercospora WDL2 described in claim 1 is cultivated（Cercospora sp.nov. WDL2）CCTCC NO：M 2014518
The method of bacterial strain, including by the Cercospora WDL2（Cercospora sp.nov. WDL2）CCTCC NO：The bacterium of M 2014518
Strain in for the culture medium for cultivating tail spore bacterium the step of cultivating.
6. the preparation method of microbial inoculum described in claim 2, comprises the following steps：By the Cercospora WDL2 described in claim 1
（Cercospora sp.nov. WDL2）CCTCC NO：The bacterial strains of M 2014518 obtain the microbial inoculum as active component.
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