CN1928075A - Strain capable of generating avermectin B component and utilization thereof - Google Patents
Strain capable of generating avermectin B component and utilization thereof Download PDFInfo
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Abstract
The present invention relates to one kind of Streptomyces avermitilis generating abamectin B1a and abamectin B2a only. The Streptomyces avermitilis has the strain numbered as X-28 and has the preservation number of CGMCC No. 1765. The present invention also relates to the application of the Streptomyces avermitilis in producing abamectin.
Description
Invention field
The present invention relates to a kind ofly only produce the deinsectization streptomycete (Streptomyces avermitilis) of B1a and B2a in the Avrmectin B component and utilize this bacterial strain to produce the method for Avrmectin.
Background of invention
Along with the raising of people's living standard and to the call of green food, biological pesticide gains great popularity in current agricultural chemicals market, and Avrmectin (avermectin is most popular and have a biological pesticide product of development potentiality in the current biological pesticide market AVM).AVM is a kind of efficient microbiotic by university in the Japanese north and the cooperative development of Merck company in 1979, it makes antibiotic drug effect improve 25 times than coeval drug effect, using dosage is reduced to μ g/kg level from mg/kg, and is that collection agricultural chemicals, veterinary drug and medicine are the new antibiotic of one.As a kind of novel biopesticide, Avrmectin has the advantage of following uniqueness: insecticidal spectrum is wide, and various insects, mite and zooparasite are all had very strong insecticidal activity; The insecticidal mechanism uniqueness do not have cross resistance with the medicament of using always, and efficiency time is long; Easily degraded in soil and water, it is residual also not have accumulation and persistence in vivo, does not pollute the environment, nuisanceless.Therefore, in occupation of increasing share, prospect for exploration is very optimistic in agricultural chemicals and veterinary drug market for Avrmectin and derivative thereof.
Avrmectin (also claiming Abamectin) is that what to be produced by deinsectization streptomycete (Streptomyces avermitilis) is a kind of 16 membered macrolide compounds.Usually, deinsectization streptomycete contains 8 similar components in the product that fermentative production obtained.These components are respectively A1a, A1b, and A2a, A2b, B1a, B1b, B2a and B2b, wherein the biological activity of B component significantly is better than the A component, especially with the B1a optimum, so industrial production is a purpose to obtain the B1a component all.In recent years, each production and research unit are all making great efforts to improve abamectin fermented productive rate.For example, Tianjin modern job technical school appoints superfine people to adopt ultraviolet ray, mutagenic compound lithium chloride, nitrosoguanidine and in conjunction with methionine(Met) means such as induce the bacterium S.avermitilis H0065 that sets out is carried out mutagenic treatment to obtain mutant strain N-3-133, wherein the B component reached for 85.3% (referring to biotechnology circular, 2005 the 4th phases).University Of Hebei adopts the ion implantation and spaceship-carried method that combines that Avrmectin generation bacterium is carried out mutagenesis, obtained the total titer height, Avrmectin that active principle B1a ratio is high is produced bacterium HY365, wherein the ratio of B1a component accounted for for 65.7% (University Of Hebei's journal (from the science of heat version), 2003 03 phases).But polycomponent miscellaneous situation does not obtain basic change in the tunning.Because what these still obtained after by these strain fermentation productions is the miscellaneous Avrmectin of 8 components, and these components chemical structure each other is close, make that the extraction process of Avrmectin B1a is complicated, extract recovery rate and be difficult to improve, and make utilization ratio of raw materials reduction in the fermenting process.Therefore, with innovative ideas and technology, when improving fermentation production rate, change the composition of tunning, the ratio that active principle B1a is occupied increases considerably, and then improve to extract recovery rate and product purity, is that present Avrmectin manufacturing enterprise generally needs the technical barrier that solves.
U.S. Patent No. 5206155 and No.5369021 disclose a kind of microorganism of Avrmectin specific components and method of this component being carried out selectivity production of optionally producing.Though this bacterial strain also is only to produce B1a and two kinds of components of B2a, wherein the shared ratio of B1a component will be lower than the B2a component.
We produce bacterial classification to existing Avrmectin and transform with the method that genetic engineering and cell engineering combine, finally obtain a strain through unremitting effort and only produce B1a in the Avrmectin B component and the new strain X-28 of B2a, and wherein B1a component proportion accounts for more than 50% of B component total amount greater than B2a.Like this, because the biosynthesizing direction ratio is more concentrated, help improving raw material availability and fermentation production rate.In addition, as mentioned above, the Avrmectin that common deinsectization streptomycete produces contains 8 components of similar, to therefrom extract the highest active and B1a component that toxicity is minimum, just must be in the industrial production through steps such as organic solvent extracting, crystallization and column chromatography repeatedly, complex process, cost is higher, and extraction yield is very low.And the X-28 bacterial strain among the present invention only has B1a and B2a through the Avrmectin component that fermentation produces, owing to there is not the component of other 6 kinds of similar, and the B1a ingredients constitute is more than 50%, just can obtain the B1a component so can only saltout when extracting by simple organic solvent lixiviate and a few step water, simplified the extraction process of B1a component greatly, reduce cost, and improved the extraction recovery rate.Certainly, remaining B2a component also can be used as the raw material of semi-synthetic MK-397 and ivermectin, thereby produces comprehensive utilization value.Through secular a large amount of experimental study, we also find out the optimised process that a cover is suitable for suitability for industrialized production at this new bacterial strain, and the production level of B1a has been reached more than the 3400 μ g/ml.Because this bacterial strain fully shows the very high production potential that it had and the genetic stability of component, therefore at home further exploitation make it progressively to promote production application and have very high value.
Summary of the invention
The present invention relates to the new strain X-28 of a kind of deinsectization streptomycete (Streptomyces avermitilis), this bacterial strain only produces B1a and the B2a in the Avrmectin B component, and wherein B1a component proportion accounts for more than 50% of B component total amount greater than the B2a component.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on July 21st, 2006, and deposit number is CGMCCNo.1765.
The X-28 bacterial strain is with existing 8 kinds of component (A1a of production, A1b, A2a, A2b, B1a, B1b, B2a and B2b) Avrmectin to produce bacterium GY115 (China Agricultural University is so kind as to give) be starting strain, ultraviolet ray by routine or N-methyl-N '-nitro-N-nitrosoguanidine or ethylmethane sulfonate are handled, and cultivate containing on the microbiotic separating plate substratum of Streptomycin sulphate or gentamicin or rifomycin or paraxin or its combination, pick out the single bacterium colony of resistance.Again this resistance bacterium colony dibbling is carried out to the fermention medium flat board two be commissioned to train foster.Take off bacterium colony with punch tool, extract after HPLC analyzes output and the component situation of its Avrmectin B1a, obtain the bacterium colony of only producing B component and high yield thus through confirming with solvents such as acetone or methyl alcohol." dibbling " described herein refers to inoculating needle and taps single bacterium colony on the separating plate lightly, puts on the dull and stereotyped specified location of fermentation again, and do corresponding mark on two flat boards.Separating plate temporarily keeps, after the flat-plate bacterial colony measurement result of fermenting is by the time come out, with the ferment separating plate colony inoculation inclined-plane of dull and stereotyped high yield bacterium colony of correspondence.
In addition, in order to obtain to be used for the bacterial strain of suitability for industrialized production, in selection by mutation, mutagenic obtained bacterial strain has also been carried out the shake-flask culture test, screening can promote carbon source, nitrogenous source, trace element and other somatomedins of this strain growth emphatically, and the best of breed of these nutritive ingredients.The starting point of screening is to accelerate the speed of growth of this bacterial strain as far as possible, shortens growth cycle.For well-grown bacterial strain, then further carry out shake flask fermentation test, obtain its mycelium, after extracting, solvent utilizes the wherein content of Avrmectin component situation and B1a of HPLC assay determination.Filter out thus high yield and the stable bacterial strain of Avrmectin component, carry out preservation to it.
X-28 bacterial strain of the present invention can utilize various carbon sources, comprises glucose, sucrose, Zulkovsky starch, wheat-flour, yellow vaccine powder, yam starch etc.In spawn culture and screening process, preferred carbon source is a glucose, and during the fermentation, comprises seed culture and fermentation culture, and preferred carbon source is a W-Gum.
X-28 bacterial strain of the present invention can utilize various nitrogenous sources, comprises ammonium sulfate, peptone, yeast powder, yeast extract paste, analysis for soybean powder, groundnut meal, extractum carnis, vinasse etc.In culture of strains and screening process, preferred nitrogenous source is an extractum carnis, and during the fermentation, comprises seed culture and fermentation culture, and preferred nitrogenous source is soybean cake powder, groundnut meal, yeast extract paste or its combination.
The invention still further relates to deinsectization streptomycete X-28, promptly deposit number is the method that the bacterial strain of CGMCC No.1765 carries out rejuvenation.Find that in test deinsectization streptomycete X-28 subculture of the present invention can change on the form of back several times, mainly contains five kinds of forms: the I type, the squirrel look, big and flat, the wheel shape, quantity is few; The II type, Slate grey, big and full, straw hat type, quantity is more; The III type, Slate grey, big and full, white point is arranged, quantity is few than the I type; The IV type, white colony, quantity is few; V-type, the baldness type, little, quantity is few.For the stability of the production that guarantees bacterial strain, need constantly carry out rejuvenation to bacterial strain, and verify to determine to be actually used in by the secondary of tiring and produce used bacterial strain.
The invention still further relates to and utilize deinsectization streptomycete X-28, promptly deposit number is the method for the bacterial strain industrial production Avrmectin B component of CGMCC No.1765.Comprise:
1, the eggplant bottle slant culture of the bacterial classification that will tire through the height of the secondary checking of tiring is inoculated into seed and shakes in the bottle, cultivates 60 hours for 28 ± 0.5 ℃.
2, will cultivate the back inoculum size of seed liquor merging and be transferred in the seeding tank, cultivate 50-70 hour for 28 ± 0.5 ℃ by 5/10000 (v/v);
3, the inoculum size of cultured seed by 7-10% is transferred in the fermentor tank, cultivated 220-260 hour for 28 ± 1 ℃;
4, the fermentor tank of finishing fermentation is put jar, enters the product extraction step.
The invention further relates to the extraction of tunning.The industrial fermentation product of X-28 bacterial strain of the present invention can also adopt multistep water salting-out process except can adopting traditional extraction method of abamectin.The fermented liquid that is about to put behind the jar obtains mycelium by Plate Filtration, and adopting volume is that the organic solvent of mycelium volume 1-10 about doubly directly carries out lixiviate to mycelium, and organic solvent can be selected from methyl alcohol, ethanol, toluene, ethyl acetate, acetone etc.Filter insoluble post precipitation and obtain vat liquor,,, B1a and B2a, particularly B1a are separated out from the vat liquor precipitation by changing the solubleness of solvent to B1a and B2a again to wherein adding the inorganic salt solution for preparing in advance.Wherein said inorganic salt solution is according to inorganic salt: water=20: 80 (w/v) is prepared, the preferred NaCl of inorganic salt.Precipitate adopts ethanol to carry out recrystallization as solvent, and wherein the alcoholic acid addition is according to precipitate: ethanol=1: 10 (w/v) calculates.Recrystallization carries out two, three times.Carry out solid-liquid separation after each crystallization.Mother liquor is concentrated the unified processing in back, can be used as the starting material that refine the pure product of B1a.Crystallization adopts the vacuum drying formula of putting to carry out drying.Total recovery reaches 75%, and wherein B1a content reaches more than 97%.
Explanation about the bacterial strain preservation
The new strain X-28 of the deinsectization streptomycete that the present invention relates to (Streptomyces avermitilis), it only produces B1a and B2a in the Avrmectin B component, and wherein B1a component proportion accounts for more than 50% of B component total amount greater than the B2a component.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on July 21st, 2006, and deposit number is CGMCC No.1765.
Description of drawings
Fig. 1: the Avrmectin high-pressure liquid phase (HPLC) that contains 8 components that deinsectization streptomycete GY115 produces is analyzed collection of illustrative plates.
The Avrmectin high-pressure liquid phase (HPLC) that only contains B1a and B2a component that Fig. 2: X-28 produces is analyzed collection of illustrative plates.
Fig. 3: existing Avrmectin is produced the operation chart that bacterium carries out mutagenesis screening.
Fig. 4: adopt multistep water salting-out process to extract the flow process demonstration of Avrmectin B1a.
Embodiment
Embodiment 1, substratum preparation
No. 1 substratum (bacterium culture medium):
A. substratum proportioning (by weight percentage):
Glucose 1.5%, DL-asparagine 0.05%, extractum carnis 0.3%, potassium primary phosphate 0.05%, agar powder 2.5%, distilled water supplies 100%, pH (before the sterilization) 7.4.
B. preparation
Take by weighing glucose earlier, put into large beaker, add little water and dissolve (distilled water), again extractum carnis is provoked to put into to add after the 100ml small beaker weighs up with electronic balance with little spoon and pour the large beaker of containing glucose after a small amount of distilled water dissolves into, and stir, then DL-asparagine, potassium primary phosphate respectively being put into a small beaker weighs up and adds a little distilled water, pour in the above-mentioned solution after heating is dissolved on being added with the electric furnace of asbestos tile, constant volume arrives used cumulative volume, transfers pH=7.4 (before the sterilization) after stirring with glass stick.
Pour in the 1000ml triangular flask after agar powder weighed up by 2.5% proportioning, then the substratum for preparing is gone in the triangular flask with the graduated cylinder packing, successively with 2 layers on cotton pad, kraft paper, after bandaging, puts into by cotton rope Autoclave, with 0.1Mpa, and 121 ℃ high pressure steam sterilization 30 minutes, treat that the substratum temperature enters when dropping to 45-50 ℃ on the Bechtop in the sterilisable chamber to go out with same pressure by aseptic technique and respectively pour the 25-30ml substratum in the plate of bacterium, can do after solidifying and separate bacterial classification and use.It is standby perhaps culture medium after sterilization to be poured in the test tube into bevel.
In addition, also above-mentioned substratum can be made eggplant bottle solid inclined-plane: the 2 gram agar powders (2.5%) of respectively packing in each eggplant bottle (250ml).Then the substratum branch eggplant bottle (each bottle respectively pack into 80ml) of packing into, put into Autoclave with tampon, 2 layers of gauze, kraft paper, cotton rope after bandaging successively again, with 0.1Mpa, 121 ℃ high pressure steam sterilization 30 minutes, when treating that the substratum temperature drops to 45-50 ℃, take out the eggplant bottle, on operator's console, put into the inclined-plane, standby after solidifying.
No. 2 substratum (laboratory ferment substratum):
A. culture medium prescription (by weight percentage):
W-Gum 12%, soybean cake powder 2.5%, yeast powder 1.0%, amylase 0.08%, lime carbonate 0.05%, ammonium sulfate 0.025%, cobalt chloride 6H
2O 0.002%, Sodium orthomolybdate 0.002%, and distilled water supplies 100%, pH (before the sterilization) 7.5.
B. compound method:
1) fermentation shake flask: the W-Gum that weighs up is liquefied adds amylase 60 ℃ the time earlier, till the color pastiness.The soybean cake powder, the yeast powder that weigh up are put into a container, thin up.Measure the quantity that needs with graduated cylinder, add various trace elements again, ammonium sulfate etc.Transfer pH=7.5, add lime carbonate at last, in the 250ml triangular flask of packing into.Every bottled 100ml fermented liquid of going into.
2) fermentation is dull and stereotyped: substratum is except that containing mentioned component, and other adds the agar powder of 2.5% (weight percent).0.1Mpa, the high pressure steam sterilization of 121 degree 30 minutes.After being cooled to 40-50 ℃, shake up down dull and stereotyped.
No. 3 substratum (fermentation seed culture medium):
A. culture medium prescription (by weight percentage)
W-Gum 2.5%, yeast powder 0.5%, soybean cake powder 0.8%, groundnut meal 1.0%, yeast extract paste 0.4%, cobalt chloride 6H
2O 0.003%, and distilled water supplies 100%, pH (before the sterilization) 7.2.
B. compound method:
Earlier the W-Gum liquefaction that weighs up, liquefy water and starch merge fully, not stratifiedly just can.The yeast powder that weighs up, soybean cake powder, groundnut meal are placed in the container thin up.Then liquefaction good W-Gum and the feed liquid in the container are measured the number that needs with graduated cylinder, in the middle of again yeast extract paste being added at last.Add cobalt chloride .6H after preparing
2O.Adjust pH=7.2, pack into then and shake bottle.Dress 40ml feed liquid in each 250ml triangular flask is put into Autoclave, with 0.1Mpa, 121 ℃ high pressure steam sterilization 30 minutes or 40 minutes after bandaging with tampon, kraft paper, cotton rope successively, it is standby to put into sterilisable chamber then.
1, mutagenic treatment
1.1, ultraviolet mutagenesis handles
With the existing deinsectization streptomycete GY115 that produces Avrmectin is starting strain, and the Avrmectin that this bacterial strain produces contains 8 kinds of components (referring to Fig. 1).At first utilize the fresh slant pore of GY115 to make monospore suspension, draw the sterile petri dish that 5ml places diameter 60mm according to the method for well known to a person skilled in the art.This flat board is placed wavelength 253.7nm, and 20cm place under the ultraviolet lamp of power 30W handled 30 seconds with the concussion of uncapping of the speed of 1370 commentaries on classics/min on 7921 type magnetic stirring apparatuss.Be transferred to then on the separating plate of No. 1 substratum that contains an amount of microbiotic (gentamicin 0.0004%w/v or paraxin 0.0008%w/v or Streptomycin sulphate 0.001%w/v), place 28 ℃ of cultivations.
1.2, ultraviolet ray is in conjunction with the mutagenic treatment of lithium chloride
1.2.1, the lithium chloride pre-treatment
The fresh spore of deinsectization streptomycete GY115 is made the monospore suspension of 0.8% lithium chloride, 28 ℃ of concussions after 4 hours, therefrom draw 5ml spore suspension place the sterile petri dish of diameter 60mm.This culture dish uncapped, and to place wavelength 253.7nm, power be 30cm place under the ultraviolet lamp of 30W, simultaneously on magnetic stirring apparatus with the speed stirring of 1370 commentariess on classics/min.Dilute immediately after 30 seconds and be transferred on the separating plate of No. 1 substratum that contains an amount of microbiotic (gentamicin 0.0004%w/v or paraxin 0.0008%w/v or Streptomycin sulphate 0.001%w/v), place 28 ℃ to cultivate down.
1.2.2, the lithium chloride aftertreatment
The fresh spore of deinsectization streptomycete GY115 is made monospore suspension, after 4 hours, therefrom draw 5ml and place aseptic culture dish 28 ℃ of concussions.With the 30cm place irradiation 30 seconds of uncapping under the ultraviolet lamp that places wavelength 253.7nm, power 30W of lid culture dish, simultaneously on magnetic stirring apparatus with the speed stirring of 1370 commentaries on classics/min.Then be placed on the separating plate of No. 1 substratum that contains an amount of microbiotic (gentamicin 0.0004%w/v or paraxin 0.0008%w/v or Streptomycin sulphate 0.001%w/v) and cultivate down at 28 ℃, wherein substratum also contains the lithium chloride of 0.8% (w/v).
2, dibbling
Tap single bacterium colony on the separating plate gently with inoculating needle, put again on the specified location of the fermentation flat board that contains No. 2 substratum, and on two flat boards, make corresponding mark.
Separating plate temporarily keeps, and after the flat-plate bacterial colony measurement result of fermenting is by the time come out, correspondence is fermented the separating plate colony inoculation of dull and stereotyped high yield bacterium colony to the inclined-plane.
3, titration
Bacterium colony is dug down together with the nutrient agar integral body of periphery and below greater than the punch tool of colony diameter with internal diameter and to place 5ml tool plug small test tube, add acetone or the methyl alcohol of 2ml, concussion 15 minutes on the earthquake device is got supernatant liquor subsequently and is carried out HPLC mensuration under the room temperature.HPLC condition determination: Kromasil C
18(200mm * 4.6mm), moving phase is methyl alcohol to reverse-phase chromatographic column: water (80: 20), flow velocity 1ml.min
-1
Specific operation process can be referring to Fig. 3.
4, result
Obtain the sudden change deinsectization streptomycete that a strain only produces B1a and B2a in the Avrmectin B component by a large amount of shaker tests, numbering dimension X-28, the form of cultivating at No. 1 culture medium flat plate of solid is Slate grey, and is big and full, straw hat type.
The HPLC of the Avrmectin product that X-28 produces analyzes and sees Fig. 2, and wherein B1a component proportion accounts for more than 50% of B component total amount greater than the B2a component.
The deinsectization streptomycete of the X-28 of being numbered of the present invention (Streptomyces avermitilis) bacterial strain is delivered China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation on July 21st, 2006, and deposit number is CGMCC No.1765.
Select top described form and be the II type, promptly Slate grey is big and full, and the single bacterium colony of the X-28 of straw hat type, is scraped with transfering loop and to be got spore by the aseptic technique rules at sterilisable chamber, puts it in the test tube that is added with sterilized water, adds the granulated glass sphere vibration, and filters with absorbent cotton.Drawing the 0.5ml bacteria suspension dilutes 5 times according to this by coubling dilution (adding the 4.5ml sterilized water).On the flat board that contains No. 1 substratum, drip 2 bacteria suspensions with the 1ml suction pipe respectively, smoothen, cultivated 11-14 days for 28 ℃ with glass spatula.The form that separation is obtained is Slate grey, and is big and full, and single bacterium colony of straw hat type carries out the secondary checking of tiring.
The checking of tiring of embodiment 4, secondary
1) preliminary identification of tiring:
, scrape with transfering loop and to get the form that embodiment 3 obtains and be Slate grey by the aseptic technique rules at sterilisable chamber, big and full, the spore on single bacterium colony surface of straw hat type, full line is drawn on (No. 1 substratum) surface in the test tube slant of getting ready, cultivates 11-14 days for 28 ± 0.5 ℃.Taking out this list bacterium colony simultaneously inserts the seed that contains No. 3 substratum and shakes bottle and carry out shaking table and cultivate shaking speed 260 commentaries on classics/min, 28 ± 0.5 ℃ of culture temperature.Change in the fermentation shake flask that contains No. 2 substratum inoculum size 5ml seed liquor/100ml fermention medium after 48 hours over to.Fermentation shake flask is cultivated on shaking table, shaking speed 260 commentaries on classics/min, and 28 ± 0.5 ℃ of culture temperature adopted HPLC method mensuration to tire on the 9th day.
Before HPLC measured, sample treatment was: get fermented liquid 7ml in 10ml tool plug test tube, centrifugal 10 minutes of 3000 commentaries on classics/min, abandoning supernatant.Add 2ml acetone, shake 2min on the vartex mixing tank, leave standstill 10min, add ethyl acetate 5ml again, concussion 2min again with the centrifugal 10min of 3000 commentaries on classics/min, gets extraction liquid, with the filtering with microporous membrane of 0.45 μ m.Accurately draw 5.0 μ l sample filtrates and carry out HPLC mensuration.HPLC condition determination: Kromasil C
18(200mm * 4.6mm), moving phase is methyl alcohol to reverse-phase chromatographic column: water (80: 20), flow velocity 1ml.min
-1
Tire if tiring of measuring is the height more than 2000 μ g/ml, the test tube slant that then keeps this list bacterium colony switching is as tiring higher bacterial classification, and prepares its checking once more of tiring.
2) checking of tiring once more:
Choose an agar block that carries disease germs kind and insert the seed that contains No. 3 substratum and shake bottle and carry out shaking table and cultivate shaking speed 260 commentaries on classics/min, 28 ± 0.5 ℃ of culture temperature from cultivate the inclined-plane corresponding to the bacterial strain test tube of tiring higher that obtains by the preliminary identification of tiring.Change in the fermentation shake flask that contains No. 2 substratum inoculum size 5ml seed liquor/100ml fermention medium after 48 hours over to.Fermentation shake flask is cultivated on shaking table, shaking speed 260 commentaries on classics/min, and 28 ± 0.5 ℃ of culture temperature, survey in the 9th day is tired.Measuring method is the same.
The result that checking obtains if tire for twice can both reach 2000 μ g/ml, then this bacterial strain is transferred to (No. 1 substratum) on the eggplant bottle slant medium, cultivate after 11-14 days for 28 ± 0.5 ℃, refrigerator is put on this inclined-plane preserved (4 ℃), as industrial ferment-seeded.In addition, can remake rejuvenation to the test tube slant bacterial classification of tiring higher and the checking of tiring, repeat above process.
The secondary industrial fermentation test of embodiment 4, X-28
1, seed tank culture
1), seed tank culture basigamy system (weight percent)
W-Gum 3.125%, hot moulding analysis for soybean powder 1.083%, groundnut meal 1.0%, yeast extract paste 0.4%, CoCl
26H
2O 0.003%, bubble enemy 0.065%, and amylase 0.0042%, water complements to 100%.
Sterilization: pH transferred to 7.6,120 ℃ of sterilizations 25 minutes before seeding tank disappeared
The back detect parameters disappears: total reducing sugar 3.0-3.2g/100ml; Reducing sugar 0.35-0.4g/100ml; Amino nitrogen 29mg/100ml; Molten phosphorus 80ug/ml; PH 6.6-6.9.
2), seed culture
Inoculum size: the agar block that takes 0.5cm * 0.5cm from the X-28 eggplant bottle solid slant culture base of the checking acquisition of tiring through secondary, being inoculated into the 500ml that contains No. 3 substratum 100ml shakes in the bottle, cultivated 60 hours down at 28 ± 0.5 ℃, shaking speed is 260 commentaries on classics/min.Cultured nutrient solution is merged, be inoculated in the seeding tank according to the inoculum size of 5/10000 (v/v).
In the seed tank culture process, 28 ± 0.5 ℃ of jar temperature, the ventilation ratio: 0-24 hour, 0.8-1.0v/v/min; 24 hours-culture transferring (access fermentor tank), 1.5-1.8v/v/min.Stir: do not stir after the inoculation, standard-sized sheet stirs after 20 hours.
The sampling frequency: before 35 hours, sampling in every 6-8 hour once.After 35 hours, sampling in per 4 hours once.Sampling in every 1-2 hour once before the culture transferring.Sample can be measured pH, mycelial concentration and microscopy mycelia form before the culture transferring.Its sample can be measured once sugar, nitrogen, the molten phosphorus of middle accident in 8 hours.(the sampling frequency can be grasped flexibly according to practical situation)
The culture transferring index:
The mycelia form: the microscopy mycelia has balling trend, mycelia Phase, pH:6.5-7.0, cell concentration 30-50%.
The seed tank culture cycle: 60 hours.
2, fermentor cultivation
1), fermentation tank culture medium (weight percent)
W-Gum 12.44%, hot moulding analysis for soybean powder 2.56%, yeast powder 1.0%, ammonium sulfate 0.021%, MnSO
4H
2O 0.0002%, yellow soda ash 0.002%, CoCl
26H
2O 0.002%, bubble enemy 0.065%, and amylase 0.065%, water complements to 100%.
Sterilization: sterilized 25 minutes for 120 ℃, pH transfers to 7.6 before disappearing.Wherein, when temperature was raised to 70-80 ℃, liquefaction 5min continued to heat up subsequently.
The back detect parameters disappears: total reducing sugar 10-13g/100ml; Reducing sugar 3-5.5g/100ml; Amino nitrogen 35mg/100ml; Molten phosphorus 90ug/ml; PH 6.6-6.9.
2), fermentation parameter control
Inoculum size 7-10%; 28 ± 1 ℃ of jar temperature; Tank pressure 0.05 ± 0.01MP; The ventilation ratio: 0-24 hour, 0.4-0.5v/v/min; 12-120 hour, 1.2v/v/min; Put jar, 1.0v/v/min in 120 hours one.
Stir: do not stir after connecing.Inoculate about 12 hours, begin when mycelia begins balling to stir mixing speed 140rpm.
When reducing to 5.8 NaOH with 30% when following, pH is adjusted to pH6.1-6.5.When reducing sugar reduces to 1.0, can add 35% glucose, the control reducing sugar is at 1.0-1.5%.
The sampling frequency: sampling in per 8 hours is once all measured pH, mycelial concentration, mycelia form, total reducing sugar, reducing sugar, ammonia nitrogen at every turn.Fermentation to the rose and begins to measure product and tire in three days, surveyed once to putting jar every day.The titration method is seen embodiment 4.
Defoaming treatment in the fermenting process: can in the middle of fermentation, add the foam that soya-bean oil control produces.During oiling repeatedly being principle on a small quantity.
Put jar: when total reducing sugar in the fermented liquid is depleted to below 2.0%, reducing sugar is depleted to below 0.8%, and the mycelia microscopy has tangible autolysis, and pH stops fermentation when rise trend is arranged, and puts jar.
Fermentation period: about 240 hours.
Through the test of secondary industrial fermentation, the final fermentation level of X-28 has reached 3400 μ g/ml, can be used for carrying out suitability for industrialized production fully.
Embodiment 5, tunning extract
Technical process can be referring to Fig. 4.The fermented liquid of putting behind the jar is obtained mycelium by Plate Filtration, adopt ethyl acetate or acetone about 5 times of volumes directly mycelium to be carried out lixiviate, extraction time is about 24 hours.Remove by filter insoluble precipitation and obtain vat liquor.Add the NaCl aqueous solution for preparing in advance, wherein NaCl to vat liquor: water=20: 80w/v.So just changed the solubleness of solvent, B1a is separated out from the vat liquor precipitation B1a and B2a.Precipitate adopts ethanol to carry out recrystallization, wherein precipitate as solvent: ethanol=1: 10w/v.Recrystallization carries out two, three times.Carry out solid-liquid separation after each crystallization.Mother liquor is concentrated the unified processing in back, can be used as the starting material that refine the pure product of B2a.Crystallization adopts vacuum drying mode to carry out drying.Total recovery reaches 75%, and wherein B1a content reaches more than 97%.
Claims (9)
1, deinsectization streptomycete Streptomyces avermitilis, its deposit number are CGMCC No.1765.
2, the application of the described deinsectization streptomycete of claim 1 in Avrmectin is produced.
3, the described application of claim 2 is characterized in that utilizing the described deinsectization streptomycete of claim 1 to carry out fermentative production.
4, the described application of claim 3, wherein fermentative production may further comprise the steps: rejuvenation of spawn, the checking of tiring, the seed shake-flask culture, seed tank culture, fermentor cultivation and product extract.
5, the described application of claim 4, wherein the used carbon source of fermentor cultivation is a W-Gum.
6, the described application of claim 4, wherein the used nitrogenous source of fermentor cultivation is soybean cake powder, groundnut meal, yeast extract paste or its combination.
7, the described application of claim 4, what product wherein extracted employing is multistep water salting-out process.
8, the described application of claim 7, multistep water salting-out process wherein comprise salt solution is added to organic solvent soaks step in the mycelium after-filtration gained filtrate.
9, the described application of claim 8, wherein said salt solution are NaCl: the solution of water=20: 80w/v.
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| CN2006101496175A CN1928075B (en) | 2006-10-12 | 2006-10-12 | Strain capable of generating avermectin B component and utilization thereof |
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| CN2006101496175A CN1928075B (en) | 2006-10-12 | 2006-10-12 | Strain capable of generating avermectin B component and utilization thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102839143A (en) * | 2012-09-25 | 2012-12-26 | 宁夏启元药业有限公司 | Breeding method of avermectin strains |
| CN103563927A (en) * | 2012-07-30 | 2014-02-12 | 王玉万 | Plant insecticide containing avermectin B2a and B1a |
| CN112760259A (en) * | 2021-01-27 | 2021-05-07 | 丽江映华生物药业有限公司 | Coenzyme Q10 strain expanding culture method |
| CN113355382A (en) * | 2021-07-20 | 2021-09-07 | 物网慧农生物科技(平原)有限公司 | Production process of abamectin |
-
2006
- 2006-10-12 CN CN2006101496175A patent/CN1928075B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103563927A (en) * | 2012-07-30 | 2014-02-12 | 王玉万 | Plant insecticide containing avermectin B2a and B1a |
| CN102839143A (en) * | 2012-09-25 | 2012-12-26 | 宁夏启元药业有限公司 | Breeding method of avermectin strains |
| CN112760259A (en) * | 2021-01-27 | 2021-05-07 | 丽江映华生物药业有限公司 | Coenzyme Q10 strain expanding culture method |
| CN113355382A (en) * | 2021-07-20 | 2021-09-07 | 物网慧农生物科技(平原)有限公司 | Production process of abamectin |
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| CN1928075B (en) | 2010-11-17 |
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