CN104161049A - Use of pestalotiopsis uvicola metabolites in preventing and treating kiwi fruit sclerotinia sclerotiorum - Google Patents
Use of pestalotiopsis uvicola metabolites in preventing and treating kiwi fruit sclerotinia sclerotiorum Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, the artemisia japonica endophytic fungi is isolated from artemisia japonica plant living body which is collected from suburb of Guiyang City Guizhou Province by endophytic fungi separation technology, by microbial taxonomic identification, the artemisia japonica endophytic fungi is named as pestalotiopsis uvicola GMH31, the strain is preserved in China Center For Type Culture Collection (CCTCC), the preservation date is June 30, 2014, and the preservation registration number is CCTCC NO:M2014303. The most striking feature of the pestalotiopsis uvicola GMH31 is that the fermentation liquor of the strain can produce active metabolites with inhibiting effects on kiwi fruit sclerotinia sclerotiorum, phytophthora capsici and other pathogenic fungi of plant fungal diseases, and the pestalotiopsis uvicola is an important biological resource in agriculture.
Description
Technical field
The present invention relates to microbial technology field, especially relating to the outstanding Wei of plan Pestalotia endogenetic fungus that a strain derives from medicinal plant Artemisia japonica can the application of plan dish stey (Pestalotiopsis uvicola) GMH31 metabolite in agricultural.
Background technology
Plant endogenesis epiphyte (fungal endophyte) refers to the certain phase of the history of life or whole stage lives and health plant is respectively organized qualified intraorganic fungi.They and host plant, by the mutual symbiotic relation closely that formed, can promote the growth of plant, and the plant adaptive capacity that reply is coerced is provided, and some plant endogenesis epiphytes also have medical value.From plant endogenesis epiphyte, find and find that reactive compound has become the another focus of domestic and international research.As Chinese patent literature CN201310194836.5 discloses separated endogenetic fungus (Metarrhizium) LD421 obtaining in a strain rough gentian, can be used for preventing and treating rough gentian leaf blight; Patent application CN201210067829.4 discloses the separated endogenetic fungus fusarium solani T-7 obtaining from the tissues such as Root of Ginkgoes, stem, leaf, and Phytophthora capsici germ, tomato wilt bacterium, Valsa mali etc. are had to good inhibitory action.
Artemisia japonica (Artemisia japonica) is composite family sagebruss, its bitter, micro-sweet, cool in nature.Among the peoplely claim again soil purple (< < Luchuan draft > >) recklessly, stomachache spirit, bear's paw careless, with all herbal medicine.There is heat-clearing, cool blood, removing toxic substances, stomach invigorating hemostasia effect, be mainly used in treating cold, fever, heatstroke, malaria, pulmonary tuberculosis hectic fever, high blood pressure, infirmities and diseases, sphincter disturbance and venomous snake bite etc.Also Artemisia japonica on the books is medicine food dual purpose plant simultaneously, contains various active material, has the different physiological roles such as immunity, antiviral, antitumor, hypoglycemic, reducing blood lipid protects the liver and delay senility.In addition, Artemisia japonica also can be used for preparing biopesticide, as insecticide, uses.
The Main Means of controlling at present crop diseases and pest crop smothering in agricultural production process is chemical pesticide control, it is to alleviating disease pest and weed, guarantee that the good harvest of crops high yield plays a positive role, but due to the mutual restriction interdependence between nature biotechnology, now do not advised too relying on chemical pesticide in the processing of carrying out disease.Therefore, the research to plant endogenesis epiphyte, utilizes the relation of plant endogenesis epiphyte and the symbiosis of host plant biologically active, screens the endogenetic fungus with high bioactivity, further obtains activated product, is a large focus of studying this year.But, for a certain host plant, which kind of endogenetic fungus or which class endogenetic fungus have resisting pathogenic microbes activity actually, and in each endogenetic fungus, which kind of bacterium activity is the highest, whether separation and purification is easy, whether fermentation process is complicated etc., all need to carry out large quantity research and be confirmed, not yet finds the concrete report of Artemisia japonica endogenetic fungus and separated cultivation thereof and resisting pathogenic microbes activity, medical active application at present.
Summary of the invention
The object of the invention is, for the deficiency that chemicals is dangerous and microbial source drug variety is less, provides a kind of endogenetic fungus separating from medicinal plant Artemisia japonica---and You Wei can plan dish stey (Pestalotiopsis uvicola).
Endogenetic fungus of the present invention, separated acquisition the medicinal plant Artemisia japonica (Artemisia japonica) from suburb, Guiyang City, Guizhou Province, called after Nei Shengyouwei can plan dish stey (Pestalotiopsis uvicola) GMH31, and the fermentating metabolism product of this bacterial strain has been carried out to multiple bioactivity research.This invention for microbial pesticide and medicine further development and exploitation good starting strain is provided.
Technical solution of the present invention is as follows:
The outstanding Wei of the present invention can plan dish stey separation, screening and evaluation:
Be in the Artemisia japonica blade gathering in suburb, Guiyang City, Guizhou Province in August, 2011, through steps such as separation, purifying, cultivation, fermentation and determinations of activity, obtain and preserve; Through microbial taxonomy, being accredited as You Wei can plan dish stey (Pestalotiopsis uvicola), name into You Wei can plan dish stey (Pestalotiopsis uvicola) GMH31.This bacterial strain is in the center preservation of Chinese Typical Representative culture collection, preservation date on June 30th, 2014, preservation registration number CCTCC NO:M2014303, Luojiashan, Wuchang, Wuhan City, Hubei Province, address.
Concrete isolated culture method is:
(1) preparation of Japanese wormwood herb botanical tissue: gather the leaf texture of Artemisia japonica, rinse with running water, the hypergene microorganism of removing sample surfaces, is then put in wind on clean gauze and dries;
(2) show sterilization and aseptic detection: the tissue block after drying is cut into suitable size, with sterile purified water, rinse well, blade sterilization adopts 75% alcohol immersion 1-2min, 0.1%HgCl
2solution soaks 3-4min, then with sterile purified water, rinses to thimerosal noresidue, with sterilizing filter paper, blots after tissue segments remained on surface water droplet, is cut into the segment of organizing of diameter 4～6mm size;
(3) separation and purification of endogenetic fungus: will organize segment to be inoculated in to contain streptomycin sulphate (100 μ gmL after step (2) sterilization
-1) and penicillin (50 μ gmL
-1) PDA culture medium flat plate on, after sealing in 25-30 ℃ of constant temperature culture 2～7 days, from tissue block, grow to mycelium, adopt most advanced and sophisticated mycelia picking method, mycelia is transferred on purifying PDA culture medium flat plate, at 25-30 ℃, carry out purifying and cultivate 5～9 days, grow complete bacterium colony, be Artemisia japonica endogenetic fungus.
Preferably, in described step (3), cultivation temperature is 28 ℃, dull and stereotyped cultivation, and purifying incubation time is 7 days.
The outstanding Wei of this Artemisia japonica endogenetic fungus called after can plan dish stey GMH31, its solid culture is characterized as: it is 9cm that bacterium colony 28 ℃ of cultivations on PDA flat board extend to diameter for 8-9 days, bacteria colony white flocculence, colyliform expansion is not obvious, the light crocus in the back side, without wheel line, fruit body prepared Chinese ink shape, particle is larger, distributes more sparse.Micro-morphology: aceravlus black, closely spherical, just bury under raw Cuticle, the ripe rear epidermis of breaking through exposes, and is a point-like; Conidium 5 cells, two teloblasts colourless (being taper shape or triangle), it is olive colour to brown (9.7～12.6 μ m) that intermediate cell is, and nearly spindle is upright, wall thickness, size is 19.7～23.5 * 5.5～6.6 μ m; Appendage and is born in conidial top, is 2～3, and angle is opened a business, and more elongated, length is 5.9～6.5 μ m, and bottom is colourless, triangle, the short or nothing of tailfiber.See Fig. 1, Fig. 2.
The molecular biological characteristics of bacterial strain GMH31: adopt molecular biology round pcr, determined dna sequence analysis, the ITSrDNA genome of bacterial strain GMH31 is comprised of 495 bases (bp).Take ITS rDNA sequence as fundamental construction phylogenetic tree, and this bacterium is that You Wei can plan dish stey, sees Fig. 3.
Another object of the present invention is to provide the method for utilizing this bacterial strain fermentation liquor metabolite control fungal diseases of plants.
Above-mentioned purpose is achieved through the following technical solutions:
A preparation method who prevents and treats the microbial metabolic products of the fungal diseases of plants such as kiwi fruit brown rot and capsicum epidemic disease, carries out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei that separation is preserved can move on potato glucose PDA solid plate by plan dish stey GMH31 bacterial classification, under 28 ± 1 ℃ of conditions, cultivate, covering whole culture dish to mycelia, is that the punching of 7mm card punch obtains bacterium cake with diameter, for inoculating;
(2) liquid fermentation and culture: fermentation culture is to contain corn flour 10g, mannitol 20g, glucose 20g, yeast extract 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 ℃ of sterilizing 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 7mm bacterium cake, under 28 ± 1 ℃ of conditions, standing for fermentation is cultivated 28-35 days;
(3) broth extraction: collect zymotic fluid after fermentation, add the ethyl acetate extraction of 1-3 times of volume, take off a layer solution, again with the extracting n-butyl alcohol of 1-2 times of volume, after layering, get upper strata liquid, under vacuum condition, be concentrated into medicinal extract, obtain n-butanol zymotic fluid extract.
A further object of the present invention is to provide this strain fermentation mycelium metabolite in the antitumor application waiting in medicine of preparation.
Above-mentioned purpose is achieved through the following technical solutions:
A preparation method with the mycelium metabolite of antitumor activity, carries out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei that separation is preserved can move on potato glucose PDA solid plate by plan dish stey GMH31 bacterial classification, under 28 ± 1 ℃ of conditions, cultivate, covering whole culture dish to mycelia, is that the punching of 7mm card punch obtains bacterium cake with diameter, for inoculating;
(2) liquid fermentation and culture: fermentation culture is to contain corn flour 10g, mannitol 20g, glucose 20g, yeast extract 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 ℃ of sterilizing 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 7mm bacterium cake, under 28 ± 1 ℃ of conditions, standing for fermentation is cultivated 28-35 days;
(3) mycelium extracts: after fermentation, collect mycelium, adding concentration is 30-90% aqueous acetone solution, make the concentration of mycelium in aqueous acetone solution be less than or equal to 0.50g/L, at power, it is ultrasonic extraction 2-5 time under 40KHz, the condition of 30min/ time, then under equal conditions add again equal-volume ethyl acetate ultrasonic extraction 2-5 time, filter, collect upper strata ethyl acetate solution, under vacuum condition, be concentrated into medicinal extract, obtain ethyl acetate mycelium extract.
Beneficial effect of the present invention:
The one, the present invention from medicinal plant Artemisia japonica blade, separate metabolite Activities of Some Plants fungal disease pathogen and tumour cell are had to strong inhibiting outstanding Wei can plan dish stey GMH31, this in agricultural, prevent and treat fungal diseases of plants pathogen and pharmaceutically the developing of anti-malignant tumor source new drugs a kind of microbial strains is provided.
The 2nd, the invention provides utilize outstanding Wei can plan dish stey GMH31 fermentation obtain the method for active-fermented broth metabolite; This metabolite can be applicable to the control of fungal diseases of plants, for the exploitation of disinfectant use in agriculture has increased new approach.
The 3rd, the invention provides utilize outstanding Wei can plan dish stey GMH31 fermentation obtain the method for active mycelium body metabolite; This metabolite can be applicable to the preparation of antineoplastic, for the new exploitation of originating of medical has increased new approach.
Accompanying drawing explanation
Fig. 1 is the lithograph that You Wei can plan dish stey (Pestalotiopsis uvicola) GMH31 bacterial strain.
Fig. 2 is the conidium figure that You Wei can plan dish stey (Pestalotiopsis uvicola) GMH31 bacterial strain.
Fig. 3 is the gene tree graph that You Wei can plan dish stey (Pestalotiopsis uvicola) GMH31 bacterial strain.
Embodiment
In order to make those of ordinary skills better understand the present invention, the applicant has carried out series of experiment research, to prove effect of the present invention.
Below, enumerate embodiment the present invention is further described, but the present invention is not limited to following embodiment.
The outstanding Wei of embodiment 1:(can plan dish stey GMH31 isolation and screening)
The present invention gathers medicinal plant---the healthy leaves of Artemisia japonica in suburb, Guiyang City, Guizhou Province in August, 2011, through surface sterilization, endogenetic fungus separation, cultivation, fermentation, determination of activity and the steps such as screening to inhibition fungal diseases of plants pathogen active bacterial strain and tumor cell line, therefrom obtaining outstanding Wei can plan dish stey GMH31, preserves.
Concrete isolated culture method is: the preparation of (1) Japanese wormwood herb botanical tissue: gather the leaf texture of Artemisia japonica, with running water, rinse, the hypergene microorganism of removing sample surfaces, is then put in wind on clean gauze and dries;
(2) show sterilization and aseptic detection: the tissue block after drying is cut into suitable size, with sterile purified water, rinse well, blade sterilization adopts 75% alcohol immersion 1min, 0.1%HgCl2 solution to soak 3min, then with sterile purified water, rinse to thimerosal noresidue, with sterilizing filter paper, blot after tissue segments remained on surface water droplet, be cut into the segment of organizing of diameter 4～6mm size;
(3) separation and purification of endogenetic fungus: will organize segment to be inoculated in to contain streptomycin sulphate (100 μ gmL after step (2) sterilization
-1) and penicillin (50 μ gmL
-1) PDA culture medium flat plate on, after sealing, in 28 ℃ of constant temperature culture 5 days, from tissue block, grow to mycelium, adopt most advanced and sophisticated mycelia picking method, mycelia is transferred on purifying PDA culture medium flat plate, at 28 ℃, carry out purifying and cultivate 7 days, grow complete bacterium colony, be Artemisia japonica endogenetic fungus.
Outstanding Wei can plan dish stey GMH31 Antibacterial Activity:
Learn from else's experience the zymotic fluid 1mL of 0.22 μ m membrane filtration in sterile petri dish, the PDA medium that is cooled to 50 ℃ with 9mL mixes rapidly, after cooling, in each medium plane, put respectively the kiwi fruit brown rot germ that 1 diameter is 7mm (Sclerotinia sclerotiorum) or phytophthora blight of pepper (Phytophthora capsici) etc. for examination bacterium bacterium cake, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 ℃ of cultivation 72h in incubator, with sterile water, process in contrast, repeat 3 times; Adopt right-angled intersection method to measure colony diameter, with following formula, calculate inhibiting rate: I (%)=[(contrast colony diameter-processing colony diameter)/(contrast colony diameter-7)] * 100%.
Measurement result shows: You Wei can be respectively 82.8% and 86.9% to the inhibiting rate of kiwi fruit brown rot germ and phytophthora blight of pepper by plan dish stey GMH31 metabolite.
Outstanding Wei can plan dish stey GMH31 inhibition tumor cell strain growth measurement:
By tumour cell suspension inoculation, in 96 well culture plates, every hole 100 μ L, after standard conditions cultivation 12h, add respectively the culture fluid (concentration is respectively 20 μ g/mL and 200 μ g/mL) that contains contrast or fermentation mycelium extract.Standard conditions cultivate respectively 12,24,48,72,96h, in cultivation, finish front 4h, and each hole adds 0.5% MTT solution 20 μ L.After cultivation finishes, abandon supernatant, every hole adds dimethyl sulfoxide (DMSO) (DMSO) 150 μ L cessation reactions, in automatic microplate reader, measure each hole in the optical density value (OD value) at wavelength 492nm place, calculate cell inhibitory rate: I (%)=[1-medicine feeding hole OD value/control wells OD value] * 100%.Inhibiting rate >50%'s is the bacterial strain with tumor cytotoxic activity.
Measurement result shows: concentration is that the outstanding Wei of 20 μ g/mL can plan dish stey GMH31 metabolite, when 72h, to Murine Ascitic Hepatoma Cells H
22, mouse hydroperitoneum type cervical cancer cell U
14with mouse hydroperitoneum type sarcoma cell S
180inhibiting rate be respectively 86.1%, 92.4% and 77.9%.
The outstanding Wei of embodiment 2:(can plan dish stey GMH31 fermentating metabolism product preparation method)
Carry out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei that separation is preserved can move on potato dextrose agar PDA solid plate by plan dish stey GMH31 bacterial classification, under 28 ± 1 ℃ of conditions, cultivate, covering whole culture dish to mycelia, is that the punching of 7mm card punch obtains bacterium cake with diameter, for inoculating;
(2) liquid fermentation and culture: fermentation culture is to contain corn flour 10g, mannitol 20g, glucose 20g, yeast extract 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 300mL triangle; Prepare rear 121 ℃ of sterilizing 20min, to be cooled to 40 ℃ of bacterium cakes that picking 1 diameter is 7mm under aseptic condition, under 28 ± 1 ℃ of conditions, standing for fermentation is cultivated 30 days;
(3) broth extraction: collect zymotic fluid after fermentation, add the extraction of equal-volume ethyl acetate, take off a layer solution, then use equal-volume extracting n-butyl alcohol, get upper strata liquid after layering, be concentrated into medicinal extract under vacuum condition, obtain n-butanol zymotic fluid extract;
(4) mycelium extracts: after fermentation, collect mycelium, adding concentration is 80% aqueous acetone solution, make the concentration of mycelium in aqueous acetone solution be less than or equal to 0.50g/L, at power, it is ultrasonic extraction 4 times under 40KHz, the condition of 30min/ time, then under equal conditions add again equal-volume ethyl acetate ultrasonic extraction 4 times, filter, collect upper strata ethyl acetate solution, under vacuum condition, be concentrated into medicinal extract, obtain ethyl acetate mycelium extract.
Outstanding Wei described in following examples 3 can plan dish stey GMH31 metabolite be n-butanol zymotic fluid extract prepared in 2 times methods of embodiment (3); The percentage composition of described product is mass/volume percentage.
The outstanding Wei of embodiment 3:(can the inhibitory action of plan dish stey GMH31 zymotic fluid metabolite to kiwi fruit brown rot germ and phytophthora blight of pepper)
(1) accurately taking You Wei can plan dish stey GMH31 n-butanol zymotic fluid extract 0.1g, adds the sterile water of 10mL volume, and ultrasonic oscillation fully dissolves, and is finally configured to the mother liquor that concentration is 10g/L;
(2) Antibacterial Activity: get above-mentioned mother liquor and be diluted to respectively the solution that concentration is 1000mg/L and 500mg/L, get each concentration solution 1mL in sterile petri dish, be cooled to the PDA medium of 50 ℃ with 9mL and mix rapidly (You Wei can plan dish stey GMH31 metabolite final concentration be 100mg/L and 50mg/L), after cooling, in each medium plane, put respectively kiwi fruit brown rot germ and the phytophthora blight of pepper bacterium cake that 1 diameter is 7mm, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 ℃ of cultivation 36h in incubator, with conventional chemical medicament and sterile water, process in contrast, repeat 3 times, adopt right-angled intersection method to measure colony diameter, with following formula, calculate inhibiting rate: I (%)=[(contrast colony diameter-processing colony diameter)/(contrast colony diameter-7)] * 100%.
(3) product of the present invention and conventional pesticide control efficiency comparative trial: result of the test is in Table 1.As seen from Table 1, You Wei is can plan dish stey GMH31 metabolite inhibition slightly excellent compared with conventional chemical medicament, and safety.
The outstanding Wei of table 1 can the inhibition of plan dish stey GMH31 metabolite to confession examination disease fungus
"-" represents not test (N.T.).
Outstanding Wei described in following examples 4 can plan dish stey GMH31 metabolite be ethyl acetate mycelium extract prepared in 2 times methods of embodiment (4); The percentage composition of described product is mass/volume percentage.
The outstanding Wei of embodiment 4:(can the inhibitory action of plan dish stey GMH31 fermentation mycelium metabolite to Murine Ascitic Hepatoma Cells H22, mouse hydroperitoneum type cervical cancer cell U14 and mouse hydroperitoneum type sarcoma cell S180)
(1) accurately taking You Wei can plan dish stey GMH31 ethyl acetate mycelium extract, after first dissolving with a small amount of ethanol, then is mixed with water for injection dilution the solution that concentration is 0.2mg/mL, for mouse, fills with and feeds administration;
(2) preparation of tumor cell line animal model: respectively Murine Ascitic Hepatoma Cells H22, mouse hydroperitoneum type cervical cancer cell U14 and mouse hydroperitoneum type sarcoma cell S180 are made to 1 * 10
6cell suspension, gets this cell suspension and only injects mouse (t is sheerly some of 615 mouse, Balb/c mouse, kunming mouse, body weight 20-22g) abdominal cavity with 0.5ml/.Be divided at random control group, experimental group next day.Every mouse stomach 1mL physiological saline of control group, the ethyl acetate mycelium extract aqueous solution 1mL that the above-mentioned concentration of every mouse stomach of experimental group is 0.2mg/mL.Gavage is 5 days continuously, observes mouse storaging current, calculates survival rate and increase in life span.
(3) the anti-tumor in vivo effect test of product of the present invention: result of the test is in Table 2.As seen from Table 2, You Wei can plan dish stey GMH31 metabolite to suppress tumor effect better, and safety.
The outstanding Wei of table 2 can plan dish stey GMH31 metabolite anti-tumor in vivo result of the test
Survival rate: observe 60 days bearing tumors not above.
The outstanding Wei of embodiment 5:(can plan dish stey GMH31 fermentation mycelium metabolite animal toxicity test)
(1) acute toxicity experience: get 40 of kunming mices (male and female half and half), body weight 22-22g, in 24 hours, gavage is 3 times, the dose that makes accumulative total is above-mentioned medicinal 225 times, Continuous Observation mouse 14 days, without the phenomena of mortality and bad reaction, illustrate that this fermentation mycelium metabolite has no side effect.
(2) long term toxicity test: get 40 of the rats (male and female half and half) that body weight is suitable, be divided at random two groups, one group was that You Wei can plan dish stey GMH31 fermentation mycelium metabolite experimental group, with 100 times of continuous gavages of above-mentioned routine test dosage 60 days; Another is organized as adding equivalent distilled water control group, found that, biped is all without death and bad reaction during this period, and two treated animal appetite, stool and urine and body weight be there was no significant difference (p>0.05) statistically; There was no significant difference (p>0.05) between two groups of routine blood test indexs such as blood leucocyte, red blood cell and clamour version; Two treated animal internal organs anatomy and pathologies observations, all without abnormal pathological phenomenon, illustrate that this fermentation mycelium metabolite long-term taking is safe and reliable.
(3) whole experimental result shows: You Wei can have direct lethal effect to tumour by plan dish stey GMH31 fermentation mycelium metabolite, and has obvious treatment function of tumor; And have no side effect, safe and reliable.
Claims (3)
1. outstanding Wei can the application of plan dish stey (Pestalotiopsis uvicola) GMH31 metabolite in control kiwi fruit brown rot germ (Sclerotinia sclerotiorum), described outstanding Wei can plan dish stey GMH31 in the center preservation of Chinese Typical Representative culture collection, preservation registration number is CCTCC NO:M2014303.
2. application as claimed in claim 1, is characterized in that: described metabolite is prepared by the following method:
(1) activation culture of bacterial classification: the outstanding Wei that separation is preserved can move on potato glucose PDA solid plate by plan dish stey GMH31 bacterial classification, under 28 ± 1 ℃ of conditions, cultivate, covering whole culture dish to mycelia, is that the punching of 7mm card punch obtains bacterium cake with diameter, for inoculating;
(2) liquid fermentation and culture: fermentation culture is to contain corn flour 10g, mannitol 20g, glucose 20g, yeast extract 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 ℃ of sterilizing 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 7mm bacterium cake, under 28 ± 1 ℃ of conditions, standing for fermentation is cultivated 28-35 days;
(3) broth extraction: collect zymotic fluid after fermentation, add the ethyl acetate extraction of 1-3 times of volume, take off a layer solution, again with the extracting n-butyl alcohol of 1-2 times of volume, after layering, get upper strata liquid, under vacuum condition, be concentrated into medicinal extract, obtain n-butanol zymotic fluid extract.
3. application as claimed in claim 1, is characterized in that: described metabolite is prepared by the following method:
(1) activation culture of bacterial classification: the outstanding Wei that separation is preserved can move on potato glucose PDA solid plate by plan dish stey GMH31 bacterial classification, under 28 ± 1 ℃ of conditions, cultivate, covering whole culture dish to mycelia, is that the punching of 7mm card punch obtains bacterium cake with diameter, for inoculating;
(2) liquid fermentation and culture: fermentation culture is to contain corn flour 10g, mannitol 20g, glucose 20g, yeast extract 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 ℃ of sterilizing 20min, to be cooled to 40 ℃ under aseptic condition picking 1 diameter be 7mm bacterium cake, under 28 ℃ of conditions, standing for fermentation is cultivated 30 days;
(3) broth extraction: collect zymotic fluid after fermentation, add isopyknic ethyl acetate extraction, take off a layer solution, then use isopyknic extracting n-butyl alcohol, get upper strata liquid after layering, be concentrated into medicinal extract under vacuum condition, obtain n-butanol zymotic fluid extract.
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