CN102154168A - Abamectin producing bacterium and preparation method thereof - Google Patents
Abamectin producing bacterium and preparation method thereof Download PDFInfo
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Abstract
The invention discloses abamectin producing bacterium and a preparation method thereof. The collection name of the bacterium is FT26-9 and the collection number of the bacterium is CGMCC No.4341. The bacterium was collected on November 12th, 2010. The preparation method comprises: performing ultraviolet and lithium chloride combined mutation induction treatment of a starting strain; screening an induced-mutation strain; performing mutation induction by nitrosoguanidine and screening; performing mutation induction by diethyl sulfate and screening; performing mutation induction by hydroxylamine and screening; performing mutation induction by 5-bromouracil and ultraviolet and screening; and finally obtaining the FT26-9 strain which can greatly improve the abamectin B component content in a fermentation product and reduce the abamectin A component content in the fermentation product. When the bacterium is used in industrial production of abamectin, the fermentation unit is improved by about 40 percent, and the product cost is reduced by about 35 percent.
Description
Technical field
The present invention relates to Avrmectin and produce bacterium and preparation method thereof, specifically Avrmectin produces variant of bacterium and preparation method thereof.
Technical background
Avrmectin (avermectin, AVM) be the class macrolide antibiotics that a class is produced through liquid fermenting by deinsectization streptomycete (streptomycesavermitilis), it is a kind of brand-new harmless boilogical agricultural and veterinary chemicals, it has high-efficiency low-toxicity and eco-friendly characteristics, relevant insect to various crop such as vegetables, fruit tree, cotton, paddy rice has good preventive effect, is the most promising in the world at present Biocidal miticide.Include 8 kinds of components (AVMA1aAVMA1b AVMA2a AVMA2b AVMB1a AVMB1b AVMB2a AVMB2b) of similar in the Avrmectin that deinsectization streptomycete fermentation at present produces.Wherein the biological activity of B component is better than the A component, and is the strongest with the B1a activity especially.Therefore how to lower the A component in the industrial fermentation product, the emphasis that raising B1a component has become people's research.For example, chemical engineering institute of University Of Tianjin pharmaceutical engineering is that people such as Li Yan rosy clouds utilize ultraviolet ray, nitrosoguanidine and in conjunction with L-Isoleucine mutagenesis means deinsectization streptomycete carried out mutagenic treatment, obtained to significantly improve the bacterial strain of B1a component output, fermentation unit improved for 12.86% (2008 the 38th volume first phases of industrial microorganism) than starting strain.CN200410009639.2 discloses a kind of method of utilizing ultraviolet ray and nitrosoguanidine mutagenesis screening to obtain the Avrmectin superior strain; CN200610149617.5 discloses the new bacterial strain that a strain only produces B1a and B2a; But the production level of technique scheme still remains further to be improved.
Summary of the invention
One of purpose of the present invention will provide a kind of new Avrmectin to produce bacterium exactly, to increase substantially Avrmectin B1a component in the tunning.
Second purpose of the present invention is exactly the preparation method that this bacterial strain will be provided.
Avrmectin provided by the present invention produces bacterium (streptomyces avermitilis), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation.The preservation name is called FT26-9, deposit number CGMCC No.4341, preservation date on November 12nd, 2010.The classification called after deinsectization streptomycete (Streptomycesavermitilis) of China Committee for Culture Collection of Microorganisms's common micro-organisms center suggestion.
Bacterial strain of the present invention (hereinafter to be referred as FT26-9) is that the contriver passes through the mutagenesis successively through ultraviolet compounded lithium chloride, nitrosoguanidine, azanol, 5-bromouracil mutagenic compound to Avid kyowamycin MF800-6 (hereinafter to be referred as initial starting strain), and then mutagenic fungi is screened acquisition.This bacterial strain can increase substantially Avrmectin B component in the tunning, reduces Avrmectin A component; It is applied to improve fermentation unit about 40% in the Avrmectin industrial production, reduces production costs about 35%.
It is as follows that the morphology of bacterial strain of the present invention and Physiology and biochemistry are learned feature:
Colony colour: beige.
Black aerobic growth: growth.
Thalline size/mm:2～6mm.
Suitable growth temperature: 27.0-28.5 ℃.
Suitable growth pH:6.0-7.5.
The thalline appearance: bacterium colony is rounded, and fold is arranged, tarnish, and the edge is neat.
Gramstaining: the positive.
Optical absorption peak/nm:245nm.
Avrmectin provided by the present invention produces the preparation method of bacterium, and it may further comprise the steps:
A, ultraviolet compounded lithium chloride mutagenic treatment:
(1) spore suspension preparation: the spore that common Avrmectin is produced bacterium is made spore suspension, and adjusting spore concentration is 10
6Individual/milliliter, in wavelength 253.7nm, carry out ultraviolet mutagenesis;
(2) mutagenic treatment: with spore suspension serial dilution behind the ultraviolet mutagenesis is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, get the diluent of 0.1 milliliter of each concentration, be coated on the plate culture medium that contains lithium chloride, cultivated 7-9 days for 28 ± 0.5 ℃.
The screening of b, mutagenic fungi:
Single bacterium colony of cultivating after the mutagenesis is forwarded in the slant medium, and corresponding dibbling is to another culture dish substratum, cultivate after 7 days for 28 ± 0.5 ℃, get single bacterium colony bacterium piece, soaked 18～24 hours with methyl alcohol, after the concussion, measure tiring of each bacterial strain leaching liquid, leave and take 10% the height bacterial strain of tiring, carry out multiple sieve, finally pick out the starting strain I of high bacterial strain that tire at last as next mutagenic compound mutagenic treatment with shake flask culture;
C, nitrosoguanidine mutagenesis, screening:
(1) spore suspension preparation: in containing the fresh test tube slant of phosphoric acid buffer, the spore of above-mentioned starting strain I is made spore suspension, adjusting spore concentration is 10
6Individual/milliliter, filter, remove most of mycelia and substratum;
(2) mutagenic treatment: above-mentioned spore suspension is added the nitrosoguanidine mother liquor, and making nitrosoguanidine content is 1.0mg/ml, behind the abundant mixing, and in 28 ± 0.5 ℃, 220rpm oscillation treatment 40-60 minute; After mutagenic treatment finishes, stop nitrosoguanidine mutagenesis, the diluent of drawing the 0.1ml different concns respectively is coated with flat board, cultivates 7-10 days for 28 ± 0.5 ℃;
(3) screening: according to the b one step process mutagenic fungi is screened, sieve is picked out the starting strain II of high bacterial strain as next mutagenic compound mutagenic treatment that tire at last eventually;
D, ethyl sulfate mutagenesis, screening:
(1) spore suspension preparation: in containing the fresh test tube slant of phosphoric acid buffer, make spore suspension with the spore of above-mentioned starting strain II, adjusting spore concentration is 10
6Individual/milliliter, filter, remove most of mycelia and substratum;
(2) mutagenic treatment: with above-mentioned spore suspension and ethyl sulfate thorough mixing, making ethyl sulfate content is 1mg/100ml, and in 28 ± 0.5 ℃, concussion is handled, and after mutagenic treatment finishes, stops Sulfothiorine mutagenesis; The diluent of drawing the 0.1ml different concns respectively is coated with flat board, cultivates 7-10 days for 28 ± 0.5 ℃;
(3) screening: according to the b one step process mutagenic fungi is screened, sieve is picked out the starting strain III of high bacterial strain as next mutagenic compound mutagenic treatment that tire at last eventually.
E, azanol mutagenesis, screening
(1) preparation of spore suspension: getting 15 milliliters of sterilized ph is 7.0 phosphoric acid buffers, pours in the cultured III bacterial strain test tube slant, and making spore concentration is 10
6The suspension of individual/milliliter.
(2) mutagenic treatment: the spore suspension for preparing is carried out gradient dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, get each concentration dilution liquid of 0.1ml, be coated on the flat board that contains azanol that has prepared, be inverted for 28 ± 0.5 ℃ and cultivated 7-9 days.
(3) screening: according to the b one step process mutagenic fungi is screened, sieve is picked out the starting strain IV of high bacterial strain as next mutagenic compound mutagenic treatment that tire at last eventually;
F, the compound ultraviolet mutagenesis of 5-bromouracil, screening:
(1) spore suspension prepares: starting strain IV spore is scraped make spore suspension, adjusting spore concentration is 10
6Individual/ml, filter, remove most of mycelia and substratum;
(2) mutagenic treatment: add 5-bromouracil in the spore suspension, making 5-bromouracil content is 1.0-1.5mg/ml, in 28 ± 0.5 ℃, handles 5-8 hour under the rotating speed 220rpm condition; The spore suspension uviolizing 35-60 second that mutagenic treatment is crossed.
(3) draw treatment solution respectively and carry out gradient dilution, draw each concentration dilution liquid of 0.1ml and be coated with flat board, cultivated 7-9 days at 28 ± 0.5 ℃.
(4) screening: according to the b one step process mutagenic fungi is screened, finally obtain the FT26-9 bacterial strain.
The bacterial strain of the inventive method preparation can increase substantially Avrmectin B component in the tunning, reduce Avrmectin A component, and it goes down to posterity and stablizes, after the bacterial strain continuous passage 15 times, its speed of growth, gramstaining result, thalli morphology size, viable bacteria body spectral absorption peak all do not have considerable change, show that bacterial strain of the present invention meets suitability for industrialized production bacterial classification needs fully.
The prescription of used substratum and preparation method are as follows in the inventive method:
The prescription of single bacterium colony substratum forms and preparation (also being the conventional substratum of Avrmectin).
Prescription (W/V): extractum carnis 0.1-0.3%, yeast extract paste 0.2-0.5%, peptone 0.5-1.0%, glucose 0.5-0.8%, ferrous sulfate 0.001% (be made into 0.1% mother liquor and add in proportion, the time spent now joins), agar powder 2.0-2.5%, surplus are distilled water.PH 7.2-7.5 cultivated 5-8 days.
Single bacterium colony substratum is preferably filled a prescription and is (for ease of statement, hereinafter to be referred as No. 1 substratum):
Beef powder (cream) 0.1%, yeast extract paste 0.2%, peptone 0.5%, glucose 0.56%, agar powder 2.5%, ferrous sulfate 0.001%, surplus are water.
Preparation method: take by weighing glucose according to above-mentioned prescription, beef powder (cream), peptone, yeast extract paste, with dissolved in distilled water and abundant mixing, add ferrous sulfate 1mL/100mL (being formulated as 0.1% solution in advance), be settled to batch volume with distilled water, dilute hydrochloric acid with 10% or 20% sodium hydroxide solution are regulated pH to 7.2, add agar powder, the liquefaction back is divided in the triangular flask of the 500mL that packs into, every bottled 300-400ml, filled in tampon, use absorbent gauze, kraft paper is wrapped successively, puts into high-pressure steam sterilizing pan, at 121 ℃, 0.1MPa condition under the sterilization 30 minutes.When temperature is reduced to 50 ℃～60 ℃, pour substratum into same sterilising conditions the went out substratum of 20ml～25ml in the culture dish of bacterium in the aseptic technique mode, lie on the worktable, treat after its cooled and solidified standby
The prescription of slant strains substratum is formed and compound method:
Prescription (W/V): extractum carnis 0.3-0.5%, peptone 0.2-0.5%, glucose 1.5-2.0%, cobalt chloride 0.001-0.003%, sal epsom 0.15-0.025%, potassium primary phosphate 0.3-0.5%, asparagine 0.05-0.07%, agar 2.0-2.5%, surplus is a distilled water.PH 7.0-7.4 cultivated 7-10 days.
The slant strains substratum is preferably filled a prescription and is (for ease of statement, hereinafter to be referred as No. 2 substratum):
Extractum carnis 0.5% peptone 0.2%, glucose 1.5%, cobalt chloride 0.003%, sal epsom 0.15%, potassium primary phosphate 0.5%, asparagine 0.05%, agar 2.5%, surplus is a distilled water.PH 7.0-7.4 cultivated 7-10 days
Preparation method: take by weighing glucose, sal epsom, potassium primary phosphate, asparagine according to above-mentioned prescription and use dissolved in distilled water respectively, cobalt chloride is formulated as the solution of 3% (g/ml) in advance, abundant then mixing, be settled to batch volume with distilled water, dilute hydrochloric acid with 10% or 20% sodium hydroxide solution are regulated pH to 7.4, add agar powder, liquefaction back branch installs in the 250mL eggplant bottle, every bottle of 70mL～80mL, filled in tampon, wrap successively with absorbent gauze, kraft paper, put into high-pressure steam sterilizing pan, sterilization is 30 minutes under 121 ℃, the condition of 0.1MPa.Sterilization finishes when treating that the substratum temperature is reduced to 50 ℃～60 ℃, sets the inclined-plane between buffering, puts between inoculation standby after solidifying.
The prescription of seed shake-flask culture base is formed and compound method:
Prescription (W/V): W-Gum 2.0-2.5%, soybean cake powder 0.5-1.0%, groundnut meal 0.8-1.2%, yeast powder 0.5%-1.0%, yeast extract paste 0.4-0.6%, cobalt chloride 0.001%-0.003%, surplus is a water.
Seed shake-flask culture base is preferably filled a prescription and is (for ease of statement, hereinafter referred to as No. 3 substratum):
W-Gum 2.5%, soybean cake powder 0.8%, groundnut meal 1%, yeast powder 0.5%, yeast extract paste 0.4%, cobalt chloride 0.003%, surplus are water.
Preparation method: take by weighing W-Gum, soybean cake powder, groundnut meal, yeast powder, yeast extract paste, cobalt chloride solution (being formulated as the solution of 3% (g/ml) in advance) according to above-mentioned prescription, the tap water that in W-Gum, adds batch volume 80%, W-Gum is liquefied, add other compositions then, accurately be settled to batch volume.Sodium hydroxide with 10% or 20% hydrochloric acid are transferred pH value to 7.2, quantitatively divides to install in the 250ml triangular flask, and every bottle of 35ml, with tampon plug mouth, gauze, kraft paper are wrapped successively, put into the Autoclave 30min that sterilizes under 121 ℃, the condition of 0.1MPa.
The prescription of fermentation shake flask substratum is formed and compound method:
Prescription (W/V): W-Gum 13-15%, soybean cake powder 2.5-3.0%, yeast powder 1-1.5%, ammonium sulfate 0.025-0.05%, lime carbonate 0.05%-1.0%, manganous sulfate 0.00025%, Sodium orthomolybdate 0.002-0.005%, cobalt chloride 0.001-0.003%, surplus is a water.
The fermentation shake flask substratum is preferably filled a prescription and is (for ease of statement, hereinafter referred to as No. 4 substratum):
W-Gum 13%, soybean cake powder 2.5%, yeast powder 1%, ammonium sulfate 0.025%, lime carbonate 0.05%, manganous sulfate 0.00025%, Sodium orthomolybdate 0.002%, cobalt chloride 0.001%, surplus are water.
Preparation method: take by weighing W-Gum according to above-mentioned prescription, soybean cake powder, yeast powder, ammonium sulfate, lime carbonate, manganous sulfate 1ml/1000ml (being formulated as 0.23% solution in advance), Sodium orthomolybdate 1ml/1000ml (being formulated as 2% solution in advance), cobalt chloride 1ml/1000ml (being formulated as 2% solution in advance) adds the tap water of 80% times of batch volume in starch, starch weight 0.05% amylase is with starch liquefacation, add bean powder then, yeast powder, ammonium sulfate, manganous sulfate, cobalt chloride, Sodium orthomolybdate, accurately be settled to batch volume, sodium hydroxide with 10% or 10% hydrochloric acid are transferred pH value to 7.5, add lime carbonate again, quantitative packing 250ml triangular flask, (limit divides rim to stir to every bottle of 40ml, prevent precipitation of calcium carbonate), with cotton padding mouth, wrap with kraft paper, put into inherent 121 ℃ of Autoclave, 0.1MPaMPa condition under the 30min that sterilizes.
The prescription of the substratum of seeding tank is formed and compound method:
Proportioning (W/V): W-Gum 2.0-2.5%, soybean cake powder 0.5-1.0%, groundnut meal 0.8-1.2%, yeast powder 0.5%-1.0%, yeast extract paste 0.4-0.6%, cobalt chloride 0.001%-0.003%, surplus are water.
The substratum of seeding tank is preferably filled a prescription and is (for ease of statement, hereinafter to be referred as No. 5 substratum):
W-Gum 2.5%, soybean cake powder 0.8%, groundnut meal 1%, yeast powder 0.5%, yeast extract paste 0.4%, cobalt chloride 0.003%, bubble enemy, 0.02% surplus is a water.
Preparation method: press 10m
3Seeding tank is counted example
Whether 1 is complete by prescription verification raw material types, and whether quantity is accurate.In seeding tank, inject 7m with hydraulic pipe
3Tap water, open to stir, successively will input such as starch, soybean cake powder, groundnut meal, yeast extract paste, bubble enemy jar in and with water blast gun with in the jar and a jar mouth rinse the micro-Cocl of weighing specified amount well
2Add in the jar, a certain amount of sodium hydroxide of weighing adds in the jar, regulates PH to 7.0-7.5.
The prescription of fermentation tank culture medium (for ease of statement, hereinafter to be referred as No. 6 substratum) is formed and compound method:
Proportioning (W/V): W-Gum 12-15%, soybean cake powder 2.5-3.0%, yeast powder 1-1.5%, ammonium sulfate 0.025-0.035%, lime carbonate 0.05-1.5%, manganous sulfate 0.0002-0.0003%, Sodium orthomolybdate 0.002-0.005%, cobalt chloride 0.002-0.003%, bubble enemy 0.02-0.06%, surplus is a water.
Fermentation tank culture medium is preferably filled a prescription and is (for ease of statement, hereinafter to be referred as No. 6 substratum):
Proportioning (W/V):
W-Gum 14%, soybean cake powder 2.5%, yeast powder 1.2%, ammonium sulfate 0.025%, lime carbonate 0.05%, manganous sulfate 0.0002%, Sodium orthomolybdate 0.002%, cobalt chloride 0.001-%, bubble enemy 0.05%, surplus is a water.
The preparation method:
In the pond that feeds intake, inject 4-5m
3Water, and open and stir, respectively raw materials such as W-Gum, soybean cake powder, yeast powder, bubble enemy are poured in the pond that feeds intake by above prescription, stir into slurries, beat to specifying in the fermentor tank, accurately take by weighing trace elements such as ammonium sulfate, manganous sulfate, Sodium orthomolybdate, cobalt chloride by prescription with pump, directly in fermentor tank inoculation mouthful adding jar, add clear water to constant volume in fermentor tank, a certain amount of sodium hydroxide of weighing adds in the jar, regulates PH to 7.3-7.8.
Description of drawings
Fig. 1 is initial starting strain (MF800-6) meta-bolites liquid-phase chromatographic analysis collection of illustrative plates, and data parameters represented among the figure sees table 1 for details.
Fig. 2 is a FT26-9 bacterial strain meta-bolites liquid-phase chromatographic analysis collection of illustrative plates, and data parameters represented among the figure sees table 2 for details.
Fig. 3 is the schematic flow sheet of the strain preparation of bacterial strain of the present invention.
Embodiment
The present invention is described in further detail below in conjunction with drawings and Examples.
With the existing Avid kyowamycin (being called for short MF800-6) of producing Avrmectin is initial starting strain, carries out according to flow process shown in Figure 3 and following method steps:
A, ultraviolet compounded lithium chloride mutagenic treatment:
(1) spore suspension preparation: get 15 milliliters of sterilized physiological saline, pour into (substratum is No. 1 substratum) in the cultured fresh test tube slant, gently the MF800-6 spore is scraped with the aseptic inoculation pin and to make spore suspension, with the blood counting chamber counting, adjusting spore concentration is 10
6Individual/milliliter.Get the 10ml spore suspension in φ 90 sterile petri dish, and adding bar magnet, place on the ultraviolet mutagenesis case magnetic stirring apparatus, in wavelength 253.7nm, ultraviolet lamp 20 centimeters of power 30W are carried out ultraviolet mutagenesis with the spore suspension for preparing, open ware lid, magnetic agitation during irradiation, the stirring while shine, irradiation time 30-60 second.(2) mutagenic treatment: with spore suspension serial dilution behind the ultraviolet mutagenesis is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, get the diluent of 0.1 milliliter of each concentration, be coated on the plate culture medium that contains lithium chloride that has prepared, scrape evenly with spreader, cultivated 7-9 days for 28 ± 0.5 ℃.
Annotate: the preparation of lithium chloride flat board: make flat board with No. 1 substratum, accurately take by weighing lithium chloride, dissolve with less water, when the culture medium after sterilization temperature is reduced to 60-70 ℃, lithium chloride is added with filtering with microporous membrane, to the concentration of lithium chloride be 0.15%, dull and stereotyped, standby after the condensation.
The screening of b, mutagenic fungi
With single bacterium colony numbering of cultivating after the mutagenesis, be forwarded in No. 2 substratum, and with the corresponding dibbling of the toothpick of the bacterium of going out to another culture dish substratum, cultivate after 7 days for 28 ± 0.5 ℃, get the bacterium piece with slightly larger in diameter in the punch tool of single colony diameter, soaked 24 hours and shook 1 minute with methyl alcohol,, eliminate 90% low liter bacterial strain with tiring of each bacterial strain leaching liquid of liquid chromatography for measuring.The inclined-plane of the corresponding higher bacterial strain correspondence of tiring carries out multiple sieve with shake flask culture, B1a with the liquid chromatogram measuring fermented liquid tires and total titer, pick out the starting strain (for statement convenient, this starting strain be called for short bacterial strain I) of high bacterial strain of tiring at last through primary dcreening operation like this, multiple sieve and last sieve eventually as next mutagenic compound mutagenic treatment.
C, nitrosoguanidine mutagenesis, screening:
(1) spore suspension preparation: get 15 milliliters of sterilized pH 6.0 phosphoric acid buffers, pour in the cultured fresh test tube slant, gently the spore of a bacterial strain I is scraped with the aseptic inoculation pin and to make spore suspension, with the blood counting chamber counting, adjusting spore concentration is 10
6Individual/milliliter.Filter, remove most of mycelia and substratum.
(2) mutagenic treatment: get the spore suspension adding 0.3ml nitrosoguanidine mother liquor that 2.70ml has prepared, making the nitrosoguanidine final concentration is 1.0mg/ml, behind the abundant mixing, and in 28 ± 0.5 ℃, 220rpm oscillation treatment 40-60 minute.After mutagenic treatment finishes, get 1 milliliter of treatment solution immediately and carry out the mutagenesis that Macrodilution stops nitrosoguanidine, draw treatment solution and carry out serial dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, with the diluent of each gradient concentration, draw 0.1ml respectively and be coated with flat board and scrape evenly with spreader, be placed in 28 ± 0.5 ℃ of incubators and cultivated 7-10 days.
Annotate: the preparation of nitrosoguanidine mother liquor: accurately take by weighing the 4mg nitrosoguanidine, add 2-3 and drip acetone (or methane amide) solution, after in water-bath, fully dissolving, add the nitrosoguanidine mother liquor that phosphoric acid buffer is made 10mg/ml.
(3) screening: according to the b one step process mutagenic fungi is screened, eventually sieve is picked out the high bacterial strain of tiring at last as the starting strain of next mutagenic compound mutagenic treatment, (convenient for statement, this starting strain is called for short bacterial strain II).
D, ethyl sulfate mutagenesis, screening:
(1) spore suspension preparation: get 20 milliliters of sterilized pH 7.0 phosphoric acid buffers, pour in the cultured fresh test tube slant, gently the spore of bacterial strain II is scraped with the aseptic inoculation pin and to make spore suspension, with the blood counting chamber counting, adjusting spore concentration is 10
6Individual/milliliter.Filter, remove most of mycelia and substratum.
(2) mutagenic treatment: draw 16ml pH 7.0 phosphoric acid buffers, 4ml spore suspension, 0.2ml ethyl sulfate thorough mixing, making the ethyl sulfate final concentration is 1%, in 28 ± 0.5 ℃, concussion was handled 30-60 minute under the rotating speed 220rpm condition, got 1 milliliter of treatment solution after the end and added 0.5 milliliter of hypo solution termination reaction.Draw 1 milliliter of treatment solution and carry out gradient dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, each concentration dilution liquid of absorption 0.1ml is coated with flat board and smoothens with spreader, cultivates 7-10 days for 28 ± 0.5 ℃.
(3) screening: according to the b one step process mutagenic fungi is screened, eventually sieve is picked out the high bacterial strain of tiring at last as the starting strain of next mutagenic compound mutagenic treatment, (convenient for statement, this starting strain is called for short bacterial strain III).
E, azanol mutagenesis, screening
(1) preparation of spore suspension: getting 15 milliliters of sterilized ph is 7.0 phosphoric acid buffers, pours in the cultured III bacterial strain test tube slant, gently spore is scraped with the aseptic inoculation pin and makes spore suspension, and with the blood counting chamber counting, adjusting spore concentration is 10
6Individual/milliliter.Filter, remove most of mycelia and substratum.
(2) mutagenic treatment: the spore suspension for preparing is carried out gradient dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, get each concentration dilution liquid of 0.1ml, be coated on the flat board that contains azanol that has prepared, cultivated 7-9 days for 28 ± 0.5 ℃.
Annotate: contain the preparation of azanol flat board: the single bacterium colony substratum temperature after subject to sterilization adds an amount of azanol when reducing to 60-70 ℃, and making azanol concentration is 0.01%-0.015% (g/ml), shakes up, and is dull and stereotyped, standby after the condensation.
(3) screening: according to the b one step process mutagenic fungi is screened, eventually sieve is picked out the high bacterial strain of tiring at last as the starting strain of next mutagenic compound mutagenic treatment, (convenient for statement, this starting strain is called for short bacterial strain IV).
F, the compound ultraviolet mutagenesis of 5-bromouracil, screening:
(1) spore suspension preparation: the spore of bacterial strain IV scraped make spore suspension, make bacteria suspension with 15ml physiological saline, spore concentration is 10
6Individual/ml, filter, remove most of mycelia and substratum.
(2) mutagenic treatment: add 5-bromouracil in the spore suspension, making 5-bromouracil content is 1.0-1.5mg/ml, in 28 ± 0.5 ℃, handles 5 hours under the rotating speed 220rpm condition; The spore suspension that to cross through the 5-bromouracil mutagenic treatment is at wavelength 253.7nm, and 20 centimeters are carried out ultraviolet mutagenesis under the ultraviolet lamp of power 30W, opens the ware lid during irradiation, opens magnetic agitation, stirs while shine, carries out the ultraviolet mutagenesis processing, irradiation time 45 seconds.
5.3.3, draw treatment solution respectively and carry out gradient dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, draw each concentration dilution liquid of 0.1ml and be coated with the dull and stereotyped spreader drawout of also using, cultivated 7-9 days for 28 ± 0.5 ℃.
(3) screening: according to the b one step process mutagenic fungi is screened, finally obtain the FT26-9 bacterial strain.
Gained FT26-9 bacterial strain is through the detection of tiring, B1a in this strain fermentation product tire (shake flask fermentation) reach about 9000ug/ml, produce bacterium with existing common Avrmectin and compare, the content of B1a has improved about 50%, and the content of B2a has improved 80-90%; Invalid component A1a, A2a reduce the mutant strain of 90-94% and 90-94% respectively.With in patent documentation the disclosed strains A VHF-66 of CN200410009639.2 compare BIa and tire and improve 72%.
A, the preparation of shaking bottle bacterial classification
According to the proportioning of No. 3 substratum and layoutprocedure preparation seed shake-flask culture base, get the cultured height 1cm on the bacterial strain inclined-plane that tires
3Left and right sides bacterium piece, aseptic technique is inoculated in seed and shakes in the bottle, seals with tampon, puts on the 200-220r/min shaking table after binding up with gauze for 2 layers, cultivates 40～50 hours for 28 ℃ ± 0.5 ℃.
B, fermentation shake flask preparation and inoculation
Proportioning and layoutprocedure preparation fermentation shake flask substratum according to No. 4 substratum, the above seed asepsis operation for preparing is moved into the seed liquor of 5-8% in each fermentation shake flask, after cotton pad seals, put on the 200-220r/min shaking table, cultivated 10-11 days for 28 ℃ ± 0.5 ℃, put bottle and tire according to embodiment 5 liquid chromatogram measuring B1a.
The seed enlarged culturing of embodiment 3 seeding tanks
The preparation of spore suspension
Get 1-4 bottle FT26-9 bacterial strain slant tube, add 50-100 milliliter sterile distilled water, scrape slant pore gently with the aseptic inoculation pin on Bechtop, inoculate syringe needle beyond the Great Wall, wrap with 12 layers of gauze and kraft paper, it is standby to put into 4 ℃ of refrigerators.
B, according to the proportioning of No. 5 substratum preparation seed culture medium, transfer pH7.0-7.5 with sodium hydroxide before the sterilization, the steam sterilizing 30min that under 121 ℃, the condition of 0.1MPa, sterilizes, when treating that temperature is reduced to 28-30 ℃, with pressure differential method spore is inserted in the seeding tank, sealing is cultivated.Culture condition: ventilation is than 1: 28 ± 0.5 ℃ of 0.6-1.2, temperature, mixing speed are that 150-300rpm. is cultured to 40-60 hour, and the sturdy branch of microscopy mycelia is many, and even dyeing does not have assorted bacterium, can change in the fermentor tank.
Embodiment 4 fermentor cultivation
According to the proportioning of No. 6 substratum preparation fermentation tank culture medium, transfer pH7.3-7.8 with sodium hydroxide before the sterilization, the steam sterilizing 30min that sterilizes under 121 ℃, the condition of 0.1MPa when treating that temperature is reduced to 28-30 ℃, changes the seeding tank seed in the fermentor tank over to and to cultivate.Culture condition: tank pressure 0.03-0.07; Ventilation is than 1-14 hour 1: 0.6-1.2,14-put jar 1: 0.3-0.6:; Temperature 27-28.5 ℃, mixing speed is about 100rpm. cycle 280-330 hour, and before rising, pH puts jar from holding mycelia, detected in the process that B1a tires in per 8 hours, total reducing sugar, reducing sugar, pH, bacterium are dense, filtering velocity, the assorted bacterium of microscopy.Heat up after the fermentation ends and put jar, enter leaching process.
Embodiment 5
Different Avrmectins produce in the fermented liquids Avrmectin B1a detection of tiring:
Plant and instrument: high-efficient liquid phase chromatogram discuss (having mutability wavelength UV-detector); Eddy mixer; 10ml tool plug centrifuge tube; Whizzer; The 50ul microsyringe; Chromatographic data processor (workstation); Chromatographic column: the 150m m * stainless steel column C18 of 4.6m m (id), 5um.
Reagent: acetone, ethyl acetate, anhydrous methanol
The high performance liquid chromatography operational condition:
Moving phase: %+ (10-15) % of chromatogram methyl alcohol+water=(80-85); Flow: 1.0ml/min; Column temperature: room temperature; Detect wavelength: 245nm;
Operation steps:
A, specimen preparation:
Accurately draw fermented liquid 5ml with the 10ml transfer pipet; place the 10ml centrifuge tube, centrifugal (3500-4500 rev/min) separated 10-15 minute, outwelled supernatant liquid; add 2ml acetone with the 2ml transfer pipet to the mycelium sediment; vibration is 1-2 minute on vibrator, shuts down 10 minutes, and 45 minutes (noting: should avoid solution outflow in the pipe during vibration) so repeatedly vibrates; add the secondary that vibrates again after the 3ml ethyl acetate with the 5ml transfer pipet; each about one minute, leave standstill then so that the supernatant liquor clarification is centrifugal.Draw supernatant liquor 50ul in a centrifuge tube with microsyringe, add 2.0ml chromatogram methyl alcohol, vibration shakes up.
The preparation of b, standard specimen
Accurately take by weighing Avrmectin standard substance 0.04 gram (being accurate to 0.0002 gram) and be dissolved in 50 milliliters of volumetric flasks, with dissolve with methanol and be settled to scale, shake up.Shift in 1 milliliter to the 10 milliliters volumetric flask with transfer pipet, with dissolve with methanol and be diluted to scale, standby.
Under these conditions, treat instrument stabilizer after, inject standard specimen solution, treat that continuous two pin Avrmectin (B1A) peak areas change relatively less than after 1.5%, again the sample solution of preparation is injected instrument, detect.
Method of calculation:
In the formula: the S---B1a peak area; The f---correction factor; 2.05---sample adds the volume of 2 ml methanol; 0.05---draw the milliliter number of sample.
Through the liquid chromatograph detection of tiring,
Table 1: starting strain MF800-6 meta-bolites liquid phase analysis contains scale.
Table 2:FT26-9 meta-bolites liquid phase analysis contains scale.
The B1a detected result of tiring in the table 3FT26 bacterial strain different fermentations time tunning.
B1a titration result in the table 4MF800 bacterial strain different fermentations time tunning.
The B1a comparing result of tiring in table 5MF800 bacterial strain and the big production tunning of FT26.
Annotate: table 3, table 4, table 5 are to be applied in the big production of fermentation to get 10 jars of its detailed correlation datas respectively.
From Fig. 1, Fig. 2, as can be seen, the peak height of effective constituent B1a, B2b is low than FT26-9 in the MF800-6 meta-bolites, peak area is also little, invalid A1a, A2a and other unknown assorted peak heights and many.Table 1, table 2 table 3, table 4, table 5 as can be seen in the MF800-6 meta-bolites relative content of main ingredient be respectively: B1a 32.09%, B2b 19.67%, A1a 8.5%, A2a 20.32%, the B1a 5355ug/ml that tires; The main ingredient relative content is respectively B1a 48.25%, B2a 35.75%, A1a 0.63%, A2a 1.64% in the FT26-9 bacterial strain meta-bolites, the B1a 8923ug/ml that tires.Be the FT26-9 bacterial strain meta-bolites than the meta-bolites B1a of MF800-6 improved 50.4%, B2a has improved 81.7%, invalid component A1a has reduced by 92.6%, A2a has reduced by 91.9%.
Claims (2)
1. Avrmectin produces bacterium (streptomyces avermitilis), and the preservation name is called FT26-9, deposit number CGMCC No.4341, preservation date on November 12nd, 2010.
2. an Avrmectin produces the preparation method of bacterium, it is characterized in that it may further comprise the steps:
A, ultraviolet compounded lithium chloride mutagenic treatment:
(1) spore suspension preparation: the spore that common Avrmectin is produced bacterium is made spore suspension, and adjusting spore concentration is 10
6Individual/milliliter, in wavelength 253.7nm, carry out ultraviolet mutagenesis;
(2) mutagenic treatment: with spore suspension serial dilution behind the ultraviolet mutagenesis is 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, get the diluent of 0.1 milliliter of each concentration, be coated on the plate culture medium that contains lithium chloride, cultivated 7-9 days for 28 ± 0.5 ℃;
The screening of b, mutagenic fungi:
Single bacterium colony of cultivating after the mutagenesis is forwarded in the slant medium, and corresponding dibbling is to another culture dish substratum, cultivate after 7 days for 28 ± 0.5 ℃, get single bacterium colony bacterium piece, soaked 18～24 hours with methyl alcohol, after the concussion, measure tiring of each bacterial strain leaching liquid, leave and take 10% the height bacterial strain of tiring, carry out multiple sieve, finally pick out the starting strain I of high bacterial strain that tire at last as next mutagenic compound mutagenic treatment with shake flask culture;
C, nitrosoguanidine mutagenesis, screening:
(1) spore suspension preparation: in containing the fresh test tube slant of phosphoric acid buffer, the spore of above-mentioned starting strain I is made spore suspension, adjusting spore concentration is 10
6Individual/milliliter, filter, remove most of mycelia and substratum;
(2) mutagenic treatment: above-mentioned spore suspension is added the nitrosoguanidine mother liquor, and making nitrosoguanidine content is 1.0mg/ml, behind the abundant mixing, and in 28 ± 0.5 ℃, 220rpm oscillation treatment 40-60 minute; After mutagenic treatment finishes, stop nitrosoguanidine mutagenesis, the diluent of drawing the 0.1ml different concns respectively is coated with flat board, cultivates 7-10 days for 28 ± 0.5 ℃;
(3) screening: according to the b one step process mutagenic fungi is screened, sieve is picked out the starting strain II of high bacterial strain as next mutagenic compound mutagenic treatment that tire at last eventually;
D, ethyl sulfate mutagenesis, screening:
(1) spore suspension preparation: in containing the fresh test tube slant of phosphoric acid buffer, make spore suspension with the spore of above-mentioned starting strain II, adjusting spore concentration is 10
6Individual/milliliter, filter, remove most of mycelia and substratum;
(2) mutagenic treatment: with above-mentioned spore suspension and ethyl sulfate thorough mixing, making ethyl sulfate content is 1mg/100ml, and in 28 ± 0.5 ℃, concussion is handled, and after mutagenic treatment finishes, stops Sulfothiorine mutagenesis; The diluent of drawing the 0.1ml different concns respectively is coated with flat board, cultivates 7-10 days for 28 ± 0.5 ℃;
(3) screening: according to the b one step process mutagenic fungi is screened, sieve is picked out the starting strain III of high bacterial strain as next mutagenic compound mutagenic treatment that tire at last eventually;
E, azanol mutagenesis, screening
(1) preparation of spore suspension: getting 15 milliliters of sterilized ph is 7.0 phosphoric acid buffers, pours in the cultured III bacterial strain test tube slant, gently spore is scraped with the aseptic inoculation pin and makes spore suspension, and with the blood counting chamber counting, adjusting spore concentration is 10
6Individual/milliliter.Filter, remove most of mycelia and substratum;
(2) mutagenic treatment: the spore suspension for preparing is carried out gradient dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, get each concentration dilution liquid of 0.1ml, be coated on the flat board that contains azanol that has prepared, cultivated 7-9 days for 28 ± 0.5 ℃;
(3) screening: according to the b one step process mutagenic fungi is screened, sieve is picked out the starting strain IV of high bacterial strain as next mutagenic compound mutagenic treatment that tire at last eventually;
F, the compound ultraviolet mutagenesis of 5-bromouracil, screening:
(1) spore suspension prepares: starting strain IV spore is scraped make spore suspension, adjusting spore concentration is 10
6Individual/ml, filter, remove most of mycelia and substratum;
(2) mutagenic treatment: add 5-bromouracil in the spore suspension, making 5-bromouracil content is 1.0-1.5mg/ml, in 28 ± 0.5 ℃, handles 5-8 hour under the rotating speed 220rpm condition; The spore suspension uviolizing 35-60 second that mutagenic treatment is crossed;
(3) draw treatment solution respectively and carry out gradient dilution, draw each concentration dilution liquid of 0.1ml and be coated with flat board, cultivated 7-9 days at 28 ± 0.5 ℃;
(4) screening: according to the b one step process mutagenic fungi is screened, finally obtain the FT26-9 bacterial strain.
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| CN102732455A (en) * | 2012-06-05 | 2012-10-17 | 山东省科学院生物研究所 | Avermectin pesticide residual degrading bacterium, and microbial inoculum produced thereby |
| CN103834594A (en) * | 2014-03-06 | 2014-06-04 | 成都大学 | Epsilon-polylysine bacterium and preparation and fermentation method thereof |
| CN103882080A (en) * | 2014-03-17 | 2014-06-25 | 中国科学院成都生物研究所 | Effective method for preparing avermectin |
| CN105462955A (en) * | 2015-06-06 | 2016-04-06 | 扬州工业职业技术学院 | Efficient selective breeding method of biological flocculant producing strain |
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| CN106609288A (en) * | 2015-10-21 | 2017-05-03 | 上海国佳生化工程技术研究中心有限公司 | Method used for increasing industrial avermectin B<1a> yield via optimization of fermentation medium |
| CN109868239A (en) * | 2019-03-04 | 2019-06-11 | 内蒙古新威远生物化工有限公司 | A kind of avermectin bacterial strain and its screening technique |
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| CN102732455A (en) * | 2012-06-05 | 2012-10-17 | 山东省科学院生物研究所 | Avermectin pesticide residual degrading bacterium, and microbial inoculum produced thereby |
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| CN103834594A (en) * | 2014-03-06 | 2014-06-04 | 成都大学 | Epsilon-polylysine bacterium and preparation and fermentation method thereof |
| CN103882080A (en) * | 2014-03-17 | 2014-06-25 | 中国科学院成都生物研究所 | Effective method for preparing avermectin |
| CN105624142A (en) * | 2014-10-30 | 2016-06-01 | 牡丹江佰佳信生物科技有限公司 | Method for improving saccharopolyspora spinosa spinosad fermentation yield |
| CN105462955A (en) * | 2015-06-06 | 2016-04-06 | 扬州工业职业技术学院 | Efficient selective breeding method of biological flocculant producing strain |
| CN106609288A (en) * | 2015-10-21 | 2017-05-03 | 上海国佳生化工程技术研究中心有限公司 | Method used for increasing industrial avermectin B<1a> yield via optimization of fermentation medium |
| CN106609288B (en) * | 2015-10-21 | 2020-11-13 | 上海国佳生化工程技术研究中心有限公司 | Method for improving industrial abamectin B by optimizing fermentation medium1aMethod of production of |
| CN109868239A (en) * | 2019-03-04 | 2019-06-11 | 内蒙古新威远生物化工有限公司 | A kind of avermectin bacterial strain and its screening technique |
| CN113405882A (en) * | 2021-06-18 | 2021-09-17 | 大庆志飞生物化工有限公司 | Abamectin staining agent and preparation and tabletting method thereof |
| CN113405882B (en) * | 2021-06-18 | 2023-06-02 | 大庆志飞生物化工有限公司 | Avermectin coloring agent, preparation and tabletting method thereof |
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