CN101899402A - Preparation method of iturin A biofungicide - Google Patents
Preparation method of iturin A biofungicide Download PDFInfo
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Abstract
The invention relates to a preparation method of iturin A biofungicide, comprising: (1) putting bacillus subtilis trophosome into acridine orange solution to obtain a mutant strain; (2) mixing soya bean meal with tap water, heating and filtering; (3) adding glucose, peptone, monopotassium phosphate and magnesium sulphate into filtrate; adding corn starch, yeast powder and tap water after stirring and dissolving; adjusting the pH value of the solution by sodium hydroxide; adding calcium carbonate; splitting into a triangular flask; and heating to 120-125 DEG C for sterilizing; (4) carrying out rounding-slope lawn inoculation on a mutant strain to obtain a mutant strain containing an iturin A single-component mutant strain; and (5) screening the mutant strain containing the highest content of the iturin A single-component mutant strain as a product by high-efficiency liquid phase with a drug containing plate method. The invention is suitable for preventing and curing a great quantity of plant pathogenic fungi producing spores, such as fungus caused by Blumeria graminis, Botrytis cirerea and Cladosporium fulvum; and the most normal leaf surface spray method is adopted for field prevention and curing, and prevention efficiency is 60-70%.
Description
Technical field
The present invention relates to a kind of preparation method, especially relate to a kind of preparation method of iturin A biofungicide.
Background technology
Before and after 2000, EPA (EPA) has been ratified the agriculture chemical registration of company subtilises such as Agraquest, Taensa, Gustafson, Microbio in succession.The bacterial strain that uses has: QST713, GB03, MBI600 (
Http:// www.epa.gov/pesticides/biopesticides/ingredients/index.h tm), FZB24.In Japan and China Taiwan, subtilis is commercialization also, commodity Botokiller by name and " Taiwan treasured ".In December, 2003～2004 year June, domestic also have Kunming to irrigate continuous heavy rain biotech firm, Yunnan star credit biological factory, Jiangsu Su Kenongization company, Wuhan Tian Hui biotech firm, Zhejiang 5 companies of Zhe Feng kind clothing agent company to have finished the medicine card and registered temporarily, be used for disease controls such as notoginseng root rot, black shank, rice sheath blight disease, grey mould fruit rot of strawberry.
The part scholar thinks that the important mechanism of subtilis sterilizing disease-preventing is that gemma was grown surely after the living spores in the preparation was sprayed on the crop on plant both at home and abroad, reaches pathogenic bacteria with pathogenic bacteria contention nutrition and activated plant self-defence mechanism and withers away.This kind suggestion has obtained the support of China The Institute for the Control of Agrochemicals of the Ministry of Agriculture,PRC.Therefore, domestic is main weight standard by the interim preparation of registering of agricultural chemicals card with the living spores number at present.
Further investigation shows that some bacterial strain can be secreted lipopeptid class fungicidal metabolite in this bacterial classification to scientists to subtilis diseases prevention sterilization mechanism.Nineteen sixty-five, reported first such as Delcambe iturin be main active substance.Iturin is small molecule lipopeptid class material (molecular weight is dalton more than 1000), form by 7 amino acid and 1 beta-amino lipid acid, comprising iraq subtilis actinomycin A, B, C, D, E and gemma rhzomorph D, (Besson F such as F, L, Michel G, Isolation and characterization of new iturins:iturin D and iturin E, Journal ofAntibiotics, 1987,40:437-442.).Some bacterial strain can produce multiple iturin, and wherein big with the iraq subtilis actinomycin A secretory volume, anti-mycotic activity is the strongest, and it directly destroys the hypha,hyphae cell walls, makes the mycelia fracture.Peypoux equals to announce in 1978 iraq subtilis actinomycin A chemical formula: C
48H
74N
12O
14, its molecular weight is 1042 dalton (Peypoux, F., Guinand, M., Michel, G., Delcambe, L.et al:Structure of iturinA, a peptidolipid antibiotic from Bacillus subtilis.Biochemistry 17:3992-3996,1978.).
The iraq subtilis actinomycin A chemical structural formula is:
Wherein: Asn is a l-asparagine; Tyr is a tyrosine; Gln is a L-glutamic acid; Pro is a proline(Pro); Ser is a Serine.
Some bacterial strain of bacillus subtilis strain also produces another kind of small molecular weight cyclic lipopeptide material, i.e. Bio-surface active element.The main difference of it and iturin is that beta-hydroxy fatty acid has replaced beta-amino lipid acid.It does not have direct fungicidal mycelia ability, but can strengthen fungal resistance (the Thimon L of iturin, Peypoux F, Maget-Dana R, et al.Interactions of bioactive lipopeptides iturin A and surfactin from Bacillus subtilis, Biotechnol.Appl.Biochem., 1992,16:144-151.)
Academy of agricultural sciences, Shanghai ecological environmental protection institute has begun to carry out the research of genus bacillus controlling plant diseases from 1997, screen the subtilis G3 bacterial strain that produces chitinase.This strains expressed goes out good diseases prevention sterilizing function, and first its antimycotic material and sterilization mechanism is furtherd investigate at home, confirms that G3 bacterial strain excretory Fungicidal active substance has iraq subtilis actinomycin A, Bio-surface active element and chitin enzyme.Iraq subtilis actinomycin A is main Fungicidal active substance, and it directly destroys the cell walls of newborn hypha,hyphae, cell content is flowed out cause mycelia fracture, death; Bio-surfactant suppresses fungus spore germination and germ tube elongation; Chitinase suppresses fungus spore germination and germ tube elongation and suppresses the hypha,hyphae growth; The three then shows the biological effect that intensive suppresses pathogenic fungi spore germination, germ tube growth and mycelia fracture with joint efforts.
Summary of the invention
Purpose of the present invention is exactly to provide a kind of phytopathogen that is applicable to the big volume production spore of control for the defective that overcomes above-mentioned prior art existence, and preventive effect reaches the preparation method of the iturin A biofungicide about 70%.
Purpose of the present invention can be achieved through the following technical solutions:
A kind of preparation method of iturin A biofungicide is characterized in that, this method may further comprise the steps:
(1) gets the subtilis nourishing body of growth phase, be placed on acridine orange solution 24-36h, obtain mutant strain;
(2) with bean cake powder and tap water by weight 1: (70-80) mix, reheat filters then to 90-100 ℃, adds tap water in the filter residue that obtains again, continues to filter 1-2 time;
(3) in filtrate filtered, add glucose, peptone, potassium primary phosphate, sal epsom, stirring and dissolving again to wherein adding W-Gum, yeast powder and tap water, mixes obtaining solution, regulator solution pH value is 7.2-7.4, add lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, filled in bottle stopper, triangular flask is heated to 120-125 ℃, and sterilization 20-40min obtains nutrient solution;
(4) controlled temperature is 30-32 ℃, and stirring velocity is 180-200rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation in nutrient solution, and inoculation time is 60-80h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) adopt the efficient liquid phase chromatographic analysis instrument from above-mentioned bacterial strains, to filter out 6-8 the highest bacterial strain of iraq subtilis actinomycin A single component;
(6) adopt the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, filter out 1-2 fungicidal activity bacterial strain, be product.
The weight concentration of the acridine orange solution in the described step (1) is 200-300 μ g/ml.
The double gauze press filtration is adopted in filtration in the described step (2).
The weight ratio of glucose, peptone, potassium primary phosphate, sal epsom, W-Gum, yeast powder, tap water and lime carbonate add-on and bean cake powder is (0.3-0.4) in the described step (3): (0.25-0.35): (0.04-0.07): (0.02-0.04): (1.2-1.8): (0.3-0.4): (350-450): (0.2-0.25): 4.
Regulator solution pH value adopts sodium hydroxide solution in the described step (3).
The volumetric molar concentration of sodium hydroxide solution is 5-7mol/L in the described step (3).
Bottle stopper in the described step (3) is made of 1 layer of flannelette or 4-6 layer gauze.
Compared with prior art, the present invention is specially adapted to prevent and treat the plant pathogenic fungi of big volume production spore, as the microbial fungal disease of white powder germ, ash arrhizus bacteria and leaf mold, 500～600 times of liquid of 1.5% iraq subtilis actinomycin A preparation adopt the most conventional foliar spray method to carry out field control, preventive effect 60～70%.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.
Embodiment 1
(1) get the subtilis nourishing body of growth phase, being placed on weight concentration is 200 μ g/ml acridine orange solution 24h, obtains mutant strain;
(2) 4kg bean cake powder and 300ml tap water are mixed, be heated to 100 ℃, adopt the double gauze press filtration then, the filter residue after filtering adds the tap water of 300ml again, continues to use double gauze press filtration 2 times;
(3) in filtrate filtered, add 0.36kg glucose, 0.32kg peptone, 0.06kg potassium primary phosphate, 0.03kg sal epsom, open to stir and make its dissolving, after treating to dissolve fully, again to wherein adding 1.6kg W-Gum and 0.32kg yeast powder, replenish tap water 400ml, mix obtaining solution, utilizing the sodium hydroxide solution regulator solution pH value of 6mol/L is 7.3, adds 0.24kg lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, constantly shake during bottling, the substratum homogeneous in guaranteeing every bottle, utilize 4 layers of gauze as bottle stopper, the kraft paper tying is heated to 121 ℃ with triangular flask, sterilization 30min;
(4) controlled temperature is 31 ℃, and stirring velocity is 200rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation, and the shaking flask inoculation kind time is 72h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) above-mentioned bacterial strains being utilized the salt acid for adjusting pH value is 2, obtain acidic precipitation, utilize silica gel column chromatography and thin plate chromatography that it is purified, obtain crude product, and then utilize high performance liquid phase to be further purified, obtain the higher bacterial strain of purity,, filter out 7 the highest bacterial strains of iraq subtilis actinomycin A single component through high performance liquid phase (HPLC) test;
(6) utilize the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, above-mentioned bacterial strains is quantitatively added in the PDA substratum, make the pastille plate, the aseptic filter paper sheet that to be stained with leaf mycete spore suspension diameter 6mm then is affixed on the PDA media surface, after cultivating 3d under 25 ℃, observe, measure colony diameter, the concentration that no leaf mycete grows on the scraps of paper is represented with μ L/mL for the minimal inhibitory concentration (MIC) of this shake-flask culture liquid; With each colony diameter of vernier caliper measurement, sample concentration is in the log value, and the bacterium colony mean diameter transfers antibacterial probit value to, calculates the regression equation of dose concentration and antibacterial probit value, obtains effective Mlc (EC
50), utilize this method to filter out 2 the highest bacterial strains of fungicidal activity, be product.
The iturin A biofungicide that makes the results are shown in Table 1 to the botrytis cinerea estimation of biological potency.Its minimal inhibitory concentration MIC is 20.0 μ g/mL; Its concentration logarithmic value and mycelial growth inhibition rate machine value regression equation: Y=3.82+1.96X; R=0.970; Concentration EC in effectively antibacterial
50Be 4.00 μ g/mL, 95% fiducial limit is 3.13～5.13 μ g/mL.
Table 1 iturin A biofungicide is to botrytis cinerea indoor bioassay result
Annotate: inoculation bacterium cake diameter 5.00mm, bacteriostasis rate (%) subtracts 5mm calculating to measure colony diameter.Colony diameter 5.00mm represents, bacterium cake mycelium germination but can not ride on the pastille substratum is grown, and is 100% bacteriostasis rate.
Embodiment 2
(1) get the subtilis nourishing body of growth phase, being placed on weight concentration is 220 μ g/ml acridine orange solution 36h, obtains mutant strain;
(2) 4kg bean cake powder and 290ml tap water are mixed, be heated to 95 ℃, adopt the double gauze press filtration then, the filter residue after filtering adds the tap water of 300ml again, continues to use double gauze press filtration 2 times;
(3) in filtrate filtered, add 0.33kg glucose, 0.31kg peptone, 0.05kg potassium primary phosphate, 0.03kg sal epsom, open to stir and make its dissolving, after treating to dissolve fully, again to wherein adding 1.5kg W-Gum and 0.32kg yeast powder, replenish tap water 500ml, mix obtaining solution, utilizing the sodium hydroxide solution regulator solution pH value of 5mol/L is 7.2, adds 0.24kg lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, constantly shake during bottling, the substratum homogeneous in guaranteeing every bottle, utilize 4 layers of gauze as bottle stopper, the kraft paper tying is heated to 120 ℃ with triangular flask, sterilization 30min;
(4) controlled temperature is 30 ℃, and stirring velocity is 200rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation, and the shaking flask inoculation kind time is 72h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) above-mentioned bacterial strains being utilized the salt acid for adjusting pH value is 2, obtain acidic precipitation, utilize silica gel column chromatography and thin plate chromatography that it is purified, obtain crude product, and then utilize high performance liquid phase to be further purified, obtain the higher bacterial strain of purity,, filter out 7 the highest bacterial strains of iraq subtilis actinomycin A single component through high performance liquid phase (HPLC) test;
(6) utilize the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, above-mentioned bacterial strains is quantitatively added in the PDA substratum, make the pastille plate, the aseptic filter paper sheet that to be stained with leaf mycete spore suspension diameter 6mm then is affixed on the PDA media surface, after cultivating 3d under 25 ℃, observe, measure colony diameter, the concentration that no leaf mycete grows on the scraps of paper is represented with μ L/mL for the minimal inhibitory concentration (MIC) of this shake-flask culture liquid; With each colony diameter of vernier caliper measurement, sample concentration is in the log value, and the bacterium colony mean diameter transfers antibacterial probit value to, calculates the regression equation of dose concentration and antibacterial probit value, obtains effective Mlc (EC
50), utilize this method to filter out 2 the highest bacterial strains of fungicidal activity, be product.
The iraq subtilis actinomycin A assay is 1.230 μ g/ μ L in the iturin A biofungicide that makes.Its minimal inhibitory concentration MIC is 19.7 μ g/mL; Its concentration logarithmic value and mycelial growth inhibition rate machine value regression equation: Y=3.58+1.10X; R=0.996; Concentration EC in effectively antibacterial
50Be 2.39 μ g/mL, 95% fiducial limit is 2.16～2.63 μ g/mL.
Embodiment 3
The iturin A biofungicide control warmhouse booth powdery mildew of cucumber test of pesticide effectiveness:
(1) get the subtilis nourishing body of growth phase, being placed on weight concentration is 220 μ g/ml acridine orange solution 36h, obtains mutant strain;
(2) 4kg bean cake powder and 290ml tap water are mixed, be heated to 95 ℃, adopt the double gauze press filtration then, the filter residue after filtering adds the tap water of 300ml again, continues to use double gauze press filtration 2 times;
(3) in filtrate filtered, add 0.33kg glucose, 0.31kg peptone, 0.05kg potassium primary phosphate, 0.03kg sal epsom, open to stir and make its dissolving, after treating to dissolve fully, again to wherein adding 1.5kg W-Gum and 0.32kg yeast powder, replenish tap water 500ml, mix obtaining solution, utilizing the sodium hydroxide solution regulator solution pH value of 5mol/L is 7.2, adds 0.24kg lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, constantly shake during bottling, the substratum homogeneous in guaranteeing every bottle, utilize 4 layers of gauze as bottle stopper, the kraft paper tying is heated to 120 ℃ with triangular flask, sterilization 30min;
(4) controlled temperature is 30 ℃, and stirring velocity is 200rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation, and the shaking flask inoculation kind time is 72h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) above-mentioned bacterial strains being utilized the salt acid for adjusting pH value is 2, obtain acidic precipitation, utilize silica gel column chromatography and thin plate chromatography that it is purified, obtain crude product, and then utilize high performance liquid phase to be further purified, obtain the higher bacterial strain of purity,, filter out 7 the highest bacterial strains of iraq subtilis actinomycin A single component through high performance liquid phase (HPLC) test;
(6) utilize the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, above-mentioned bacterial strains is quantitatively added in the PDA substratum, make the pastille plate, the aseptic filter paper sheet that to be stained with leaf mycete spore suspension diameter 6mm then is affixed on the PDA media surface, after cultivating 3d under 25 ℃, observe, measure colony diameter, the concentration that no leaf mycete grows on the scraps of paper is represented with μ L/mL for the minimal inhibitory concentration (MIC) of this shake-flask culture liquid; With each colony diameter of vernier caliper measurement, sample concentration is in the log value, and the bacterium colony mean diameter transfers antibacterial probit value to, calculates the regression equation of dose concentration and antibacterial probit value, obtains effective Mlc (EC
50), utilize this method to filter out 2 the highest bacterial strains of fungicidal activity, be product.
The pulvis that utilizes 20 tons of jar fermented liquid spray dryings to make, iraq subtilis actinomycin A content 20mg/g, G3 living spores 50,000,000,000/g, the iturin A biofungicide that obtains is configured to 400 times of liquid (2.25kg sterilant/hectare), 500 times of liquid (1.8kg sterilant/hectare), 600 times of liquid (1.5kg sterilant/hectare), adopt the most conventional foliar spray method to carry out field control, and farmland spray CK (clear water) compared, prevention effect to powdery mildew of cucumber is as shown in table 2, in the test, iturin A biofungicide is to the blade of cucumber, flower and fruit do not have any poisoning, do not observe influence to other biology yet, therefore, iturin A biofungicide can be used as non-harmful cucurbits powdery mildew control medicine and is used.
Table 2 iturin A biofungicide greenhouse is to the powdery mildew of cucumber prevention effect
Embodiment 4
Iturin A biofungicide is prevented and treated the leaf muld of tomato test of pesticide effectiveness:
(1) get the subtilis nourishing body of growth phase, being placed on weight concentration is 200 μ g/ml acridine orange solution 36h, obtains mutant strain;
(2) 4kg bean cake powder and 320ml tap water are mixed, be heated to 98 ℃, adopt the double gauze press filtration then, the filter residue after filtering adds the tap water of 320ml again, continues to use double gauze press filtration 2 times;
(3) in filtrate filtered, add 0.36kg glucose, 0.31kg peptone, 0.06kg potassium primary phosphate, 0.03kg sal epsom, open to stir and make its dissolving, after treating to dissolve fully, again to wherein adding 1.6kg W-Gum and 0.32kg yeast powder, replenish tap water 400ml, mix obtaining solution, utilizing the sodium hydroxide solution regulator solution pH value of 5mol/L is 7.2, adds 0.24kg lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, constantly shake during bottling, the substratum homogeneous in guaranteeing every bottle, utilize 4 layers of gauze as bottle stopper, the kraft paper tying is heated to 122 ℃ with triangular flask, sterilization 40min;
(4) controlled temperature is 30 ℃, and stirring velocity is 200rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation, and the shaking flask inoculation kind time is 80h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) above-mentioned bacterial strains being utilized the salt acid for adjusting pH value is 2, obtain acidic precipitation, utilize silica gel column chromatography and thin plate chromatography that it is purified, obtain crude product, and then utilize high performance liquid phase to be further purified, obtain the higher bacterial strain of purity,, filter out 7 the highest bacterial strains of iraq subtilis actinomycin A single component through high performance liquid phase (HPLC) test;
(6) utilize the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, above-mentioned bacterial strains is quantitatively added in the PDA substratum, make the pastille plate, the aseptic filter paper sheet that to be stained with leaf mycete spore suspension diameter 6mm then is affixed on the PDA media surface, after cultivating 3d under 25 ℃, observe, measure colony diameter, the concentration that no leaf mycete grows on the scraps of paper is represented with μ L/mL for the minimal inhibitory concentration (MIC) of this shake-flask culture liquid; With each colony diameter of vernier caliper measurement, sample concentration is in the log value, and the bacterium colony mean diameter transfers antibacterial probit value to, calculates the regression equation of dose concentration and antibacterial probit value, obtains effective Mlc (EC
50), utilize this method to filter out 2 the highest bacterial strains of fungicidal activity, be product.
The pulvis that utilizes 20 tons of jar fermented liquid spray dryings to make, iraq subtilis actinomycin A content 20mg/g, G3 living spores 50,000,000,000/g, the iturin A biofungicide that obtains is configured to 400 times of liquid (2.25kg sterilant/hectare), 500 times of liquid (1.8kg sterilant/hectare), 600 times of liquid (1.5kg sterilant/hectare), adopt the most conventional foliar spray method to carry out field control, and farmland spray CK (clear water) compared, prevention effect to leaf muld of tomato is as shown in table 3, iturin A biofungicide has the better prevention effect to leaf muld of tomato, and prevention effect and using dosage are tangible dependency.The disease index difference of 3 each sub-districts of concentration is little, shows the stability of drug effect.Therefore, iturin A biofungicide is to prevent and treat comparatively ideal medicament of leaf muld of tomato at present.Whole test phase visual observation, medicament is the same with the CK check plot to tomato plant, bud and fruit, do not have any symptom of chemical damage and produce, and applying area leaf look light green, significantly better than check plot and chemoprevention district, the fertilizer efficiency effect arranged.
Table 3 iturin A biofungicide control greenhouse tomato leaf mold effect
Embodiment 5
(1) get the subtilis nourishing body of growth phase, being placed on weight concentration is 200 μ g/ml acridine orange solution 24h, obtains mutant strain;
(2) 4kg bean cake powder and 280ml tap water are mixed, be heated to 90 ℃, adopt the double gauze press filtration then, the filter residue after filtering adds the tap water of 280ml again, continues to use double gauze press filtration 1 time;
(3) in filtrate filtered, add 0.3kg glucose, 0.25kg peptone, 0.04kg potassium primary phosphate, 0.02kg sal epsom, open to stir and make its dissolving, after treating to dissolve fully, again to wherein adding 1.2kg W-Gum and 0.3kg yeast powder, replenish tap water 450ml, mix obtaining solution, utilizing the sodium hydroxide solution regulator solution pH value of 5mol/L is 7.2, adds 0.2kg lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, constantly shake during bottling, the substratum homogeneous in guaranteeing every bottle, utilize 1 layer of flannelette as bottle stopper, the kraft paper tying is heated to 120 ℃ with triangular flask, sterilization 20min;
(4) controlled temperature is 30 ℃, and stirring velocity is 180rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation, and the shaking flask inoculation kind time is 60h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) above-mentioned bacterial strains being utilized the salt acid for adjusting pH value is 2, obtain acidic precipitation, utilize silica gel column chromatography and thin plate chromatography that it is purified, obtain crude product, and then utilize high performance liquid phase to be further purified, obtain the higher bacterial strain of purity,, filter out 6 the highest bacterial strains of iraq subtilis actinomycin A single component through high performance liquid phase (HPLC) test;
(6) utilize the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, above-mentioned bacterial strains is quantitatively added in the PDA substratum, make the pastille plate, the aseptic filter paper sheet that to be stained with leaf mycete spore suspension diameter 6mm then is affixed on the PDA media surface, after cultivating 3d under 25 ℃, observe, measure colony diameter, the concentration that no leaf mycete grows on the scraps of paper is represented with μ L/mL for the minimal inhibitory concentration (MIC) of this shake-flask culture liquid; With each colony diameter of vernier caliper measurement, sample concentration is in the log value, and the bacterium colony mean diameter transfers antibacterial probit value to, calculates the regression equation of dose concentration and antibacterial probit value, obtains effective Mlc (EC
50), utilize this method to filter out 1 the highest bacterial strain of fungicidal activity, be product.
Embodiment 6
(1) get the subtilis nourishing body of growth phase, being placed on weight concentration is 300 μ g/ml acridine orange solution 36h, obtains mutant strain;
(2) 4kg bean cake powder and 320ml tap water are mixed, be heated to 100 ℃, adopt the double gauze press filtration then, the filter residue after filtering adds the tap water of 320ml again, continues to use double gauze press filtration 2 times;
(3) in filtrate filtered, add 0.4kg glucose, 0.35kg peptone, 0.07kg potassium primary phosphate, 0.04kg sal epsom, open to stir and make its dissolving, after treating to dissolve fully, again to wherein adding 1.8kg W-Gum and 0.4kg yeast powder, replenish tap water 350ml, mix obtaining solution, utilizing the sodium hydroxide solution regulator solution pH value of 7mol/L is 7.4, adds 0.25kg lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, constantly shake during bottling, the substratum homogeneous in guaranteeing every bottle, utilize 6 layers of gauze as bottle stopper, the kraft paper tying is heated to 125 ℃ with triangular flask, sterilization 40min;
(4) controlled temperature is 32 ℃, and stirring velocity is 200rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation, and the shaking flask inoculation kind time is 80h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) above-mentioned bacterial strains being utilized the salt acid for adjusting pH value is 2, obtain acidic precipitation, utilize silica gel column chromatography and thin plate chromatography that it is purified, obtain crude product, and then utilize high performance liquid phase to be further purified, obtain the higher bacterial strain of purity,, filter out 8 the highest bacterial strains of iraq subtilis actinomycin A single component through high performance liquid phase (HPLC) test;
(6) utilize the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, above-mentioned bacterial strains is quantitatively added in the PDA substratum, make the pastille plate, the aseptic filter paper sheet that to be stained with leaf mycete spore suspension diameter 6mm then is affixed on the PDA media surface, after cultivating 3d under 25 ℃, observe, measure colony diameter, the concentration that no leaf mycete grows on the scraps of paper is represented with μ L/mL for the minimal inhibitory concentration (MIC) of this shake-flask culture liquid; With each colony diameter of vernier caliper measurement, sample concentration is in the log value, and the bacterium colony mean diameter transfers antibacterial probit value to, calculates the regression equation of dose concentration and antibacterial probit value, obtains effective Mlc (EC
50), utilize this method to filter out 2 the highest bacterial strains of fungicidal activity, be product.
Claims (7)
1. the preparation method of an iturin A biofungicide is characterized in that, this method may further comprise the steps:
(1) gets the subtilis nourishing body of growth phase, be placed on acridine orange solution 24-36h, obtain mutant strain;
(2) with bean cake powder and tap water by weight 1: (70-80) mix, reheat filters then to 90-100 ℃, adds tap water in the filter residue that obtains again, continues to filter 1-2 time;
(3) in filtrate filtered, add glucose, peptone, potassium primary phosphate, sal epsom, stirring and dissolving again to wherein adding W-Gum, yeast powder and tap water, mixes obtaining solution, regulator solution pH value is 7.2-7.4, add lime carbonate again, obtain pre-nutrient solution, pre-nutrient solution branch is packed in the triangular flask, filled in bottle stopper, triangular flask is heated to 120-125 ℃, and sterilization 20-40min obtains nutrient solution;
(4) controlled temperature is 30-32 ℃, and stirring velocity is 180-200rpm, and the mutant strain that obtains in the step (1) is carried out a ring inclined-plane lawn inoculation in nutrient solution, and inoculation time is 60-80h, obtains containing the bacterial strain of iraq subtilis actinomycin A single component;
(5) adopt the efficient liquid phase chromatographic analysis instrument from above-mentioned bacterial strains, to filter out 6-8 the highest bacterial strain of iraq subtilis actinomycin A single component;
(6) adopt the pastille Plating to measure minimal inhibitory concentration and effective Mlc of above-mentioned bacterial strains, filter out 1-2 fungicidal activity bacterial strain, be product.
2. the preparation method of a kind of iturin A biofungicide according to claim 1 is characterized in that, the weight concentration of the acridine orange solution in the described step (1) is 200-300 μ g/ml.
3. the preparation method of a kind of iturin A biofungicide according to claim 1 is characterized in that, the double gauze press filtration is adopted in the filtration in the described step (2).
4. the preparation method of a kind of iturin A biofungicide according to claim 1, it is characterized in that the weight ratio of glucose, peptone, potassium primary phosphate, sal epsom, W-Gum, yeast powder, tap water and lime carbonate add-on and bean cake powder is (0.3-0.4) in the described step (3): (0.25-0.35): (0.04-0.07): (0.02-0.04): (1.2-1.8): (0.3-0.4): (350-450): (0.2-0.25): 4.
5. the preparation method of a kind of iturin A biofungicide according to claim 1 is characterized in that, regulator solution pH value adopts sodium hydroxide solution in the described step (3).
6. the preparation method of a kind of iturin A biofungicide according to claim 5 is characterized in that, the volumetric molar concentration of sodium hydroxide solution is 5-7mol/L in the described step (3).
7. the preparation method of a kind of iturin A biofungicide according to claim 1 is characterized in that, the bottle stopper in the described step (3) is made of 1 layer of flannelette or 4-6 layer gauze.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694601A (en) * | 2013-12-30 | 2015-06-10 | 中国科学院成都生物研究所 | High-efficiency preparation method of Iturin A and homologue of Iturin A |
| CN113430245A (en) * | 2021-08-03 | 2021-09-24 | 广州百仕肽生物科技有限公司 | Quantitative determination method for iturin antibacterial activity |
-
2009
- 2009-06-01 CN CN2009100523200A patent/CN101899402A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694601A (en) * | 2013-12-30 | 2015-06-10 | 中国科学院成都生物研究所 | High-efficiency preparation method of Iturin A and homologue of Iturin A |
| CN113430245A (en) * | 2021-08-03 | 2021-09-24 | 广州百仕肽生物科技有限公司 | Quantitative determination method for iturin antibacterial activity |
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