CN109868239B - Abamectin strain and screening method thereof - Google Patents
Abamectin strain and screening method thereof Download PDFInfo
- Publication number
- CN109868239B CN109868239B CN201910160954.1A CN201910160954A CN109868239B CN 109868239 B CN109868239 B CN 109868239B CN 201910160954 A CN201910160954 A CN 201910160954A CN 109868239 B CN109868239 B CN 109868239B
- Authority
- CN
- China
- Prior art keywords
- strain
- wastewater
- plate
- fermentation
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an avermectin strain and a screening method thereof, the strain is classified and named as Streptomyces avermitilis, the classified and named Latin is named as Streptomyces avermitilis, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.17157, and the preservation date is 2019, 1 month and 10 days. The screening method comprises the steps of preparing spore suspension; carrying out composite mutagenesis on the spore suspension in a lithium chloride solution, then carrying out ultraviolet lamp irradiation mutagenesis, adding NTG into the bacterial liquid treated by ultraviolet light-lithium chloride, and terminating the reaction; plate and slant culture; shake bed cultivation and screening; finally obtaining the strain. The strain is suitable for use, residual nutrient substances and organic acid in the wastewater are utilized, but the tank-placing titer of a fermentation tank is not influenced, and the discharge amount of tap water for fermentation and plate-frame wastewater can be reduced.
Description
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to an avermectin strain and a screening method thereof.
Background
The avermectin is also called avermectin, is a group of high-efficiency, low-toxicity and broad-spectrum macrolide agricultural antibiotics with a sixteen-membered ring mechanism, and has extremely strong killing activity on endoparasites. The avermectin producing strain is streptomyces avermitilis, the strain is streptomyces avermitilis separated from soil and has no fungus or bacterial activity, the secondary metabolic compound produced by the producing strain through biological liquid culture consists of 8 components, which are named as Ala, Alb, A2a, A2B, B1a, B2a, B1B and B2B, and the basic structure of the compounds is 16-membered macrolide. The biological activity of the component B1 in the 8 components is most obvious and effective, and the compound has good application value and wide market prospect in the production of medicine, agriculture and animal husbandry.
At present, about 15 avermectins are produced in our country, and about 4000 tons of avermectins are produced annually. The average fermentation level of the abamectin at the present stage is about 5000-; meanwhile, the wastewater also contains a large amount of C sources, N sources, organic matters and metabolites, the pH value is between 4.0 and 5.5, the plate frame wastewater is treated and discharged by a sewage plant through an anaerobic/oxygen consumption system, and the cost for treating one ton of plate frame wastewater is about 35 yuan/ton, so that the operation cost of enterprises is increased, the pollution to the water environment is caused, and the waste of water resources is caused.
The invention patent application CN102154168A discloses an avermectin producing strain and a preparation method thereof, wherein the preparation method comprises the steps of carrying out ultraviolet ray composite lithium chloride mutagenesis treatment on a starting strain; screening mutagenic strains; mutagenizing nitrosoguanidine and screening; mutagenesis and screening of diethyl sulfate; mutation and screening of hydroxylamine; 5-bromouracil composite ultraviolet mutagenesis and screening; finally obtaining an FT26-9 strain, wherein the strain can greatly improve the abamectin B component in the fermentation product and reduce the abamectin A component; when the method is applied to industrial production of abamectin, the fermentation unit can be improved by about 40 percent, and the production cost can be reduced by about 35 percent. However, in the screening process, the method repeatedly utilizes mutagenesis, has carcinogenesis, is complex and tedious in process, and has adverse effects on human health and environmental protection.
The invention patent application CN102634471A discloses an abamectin B1a high-yield strain and application thereof, and lithium chloride (N) is implanted into the strain by low-energy nitrogen ions+-LiCl) composite mutagenesis is combined with ultraviolet ray-lithium chloride (UV-LiCl) composite mutagenesis, a strain with high abamectin B1a yield is obtained through primary screening and shake flask fermentation and secondary screening and is used as an initial strain of next round mutagenesis, and a target strain AVE07-N2-16515 is screened finally, so that the abamectin B1a component in a fermentation product can be greatly improved, other components are reduced, and the genetic stability is good. The strain utilizes glucose as a quick-acting carbon source and corn starch as a slow-acting carbon source, the titer of the abamectin B1a produced by fermentation in a 5L fermentation tank can reach 3048 mu g/mL, the yield is increased by 23.4 percent compared with the original starting strain AVE07, the abamectin B1a can be applied to industrial production, the fermentation unit is greatly increased, and the abamectin B has great economic application value. But the titer is lower, and the running water cannot be effectively reducedWater and frame wastewater discharge.
The invention patent application CN101659970A discloses a method for circularly treating abamectin fermentation wastewater and pleurin fermentation wastewater. The fermentation wastewater generated by producing the abamectin is used for producing the pleurin by fermentation, and the fermentation wastewater generated by producing the pleurin is used for producing the abamectin by fermentation. The invention can avoid the phenomenon that the abamectin fermentation wastewater and the pleurin fermentation wastewater are randomly discharged, achieves the effect of environmental protection, simultaneously recycles the two types of wastewater, and has high utilization value, and experiments show that the titer of a product obtained by fermenting the two types of treated wastewater is equivalent to the titer of a product obtained by fermenting natural water, the invention also reduces the production cost of enterprises, saves the environmental protection investment and the treatment cost in the aspect of wastewater treatment, and can reduce the cost by 60-70 percent by using the method to treat the wastewater compared with the traditional method. However, the titer of the culture result of the invention is low, the culture process is complicated, and the process needs to be further simplified.
At present, no abamectin strain exists in China, the screening method is safe and simple, and the abamectin strain is applicable and utilizes residual nutrient substances and organic acid in wastewater after mutagenesis and domestication of plate-frame wastewater without influencing the tank placing titer of a fermentation tank; and the discharge amount of tap water for fermentation and plate frame wastewater can be reduced.
Disclosure of Invention
In order to solve the problems, the invention provides an abamectin strain and a screening method thereof, which can be applied and utilized to residual nutrient substances and organic acid in wastewater without influencing the tank placing titer of a fermentation tank after mutagenesis and domestication of plate-frame wastewater; also can reduce 30% of discharge amount of tap water for fermentation and plate frame wastewater.
In order to solve the technical problems, the invention adopts the following technical scheme:
an avermectin strain is classified and named as Streptomyces avermitilis, the classified and named Latin is named as Streptomyces avermitilis, and has been stored in the common microorganism center of China Committee for culture Collection of microorganisms, the storage unit is CGMCC for short, the address is the institute of microbiology of China academy of sciences No. 3 of North West Lu 1 institute of North West Lu of the Chaoyang district, Beijing, the storage number is CGMCC No.17157, and the storage date is 2019, 1 month and 10 days.
The strain is streptomyces avermitilis. The single colony belongs to streptomyces griseus, the shape is aerial hypha branch, the spore is observed by an electron microscope, the spore silk begins to be compact spiral, and the spore silk is gradually loosened in the later period. The number of spores on the spore chain is generally more than 15, and the spore chain is round to elliptical and has a smooth surface.
The invention also provides a screening method of the abamectin strain, which comprises the following steps:
(1) preparation of germinated spore suspension: placing the spore slant in sterile water to obtain spore suspension, and culturing the spore suspension to obtain germinated spore suspension;
(2) complex mutagenesis of germinated spore suspension: taking out part of germinated spore suspension, adding the spore suspension into a lithium chloride solution, then placing the solution under an ultraviolet lamp for irradiation, then adding NTG (nitroglycerin) and then adding normal saline to obtain a mutagenic bacterium solution;
(3) plate and slant culture: sucking mutagenic bacteria liquid, diluting 105-106After doubling, coating the diluent on a plate culture medium containing plate-and-frame wastewater for culture, and then adding the diluent into a slant culture medium to obtain a seed solution;
(4) shaking table cultivation and screening: inoculating the seed liquid into a seed shake culture medium for culture, and then transferring into a fermentation shake culture medium for culture; and (5) screening after fermentation to obtain a strain.
Further, in the step (1), the spore suspension is cultured on a shaking table for 1.5-2h at the temperature of 28-28.5 ℃ at 100 r/min.
Further, in the step (2), the ratio of the volume of the germinated spore suspension to the sterile water in the step (1) is 1: 2.
Further, in the step (2), the mass concentration of the lithium chloride solution is 0.3-0.5%.
Further, in the step (2), the taken germinated spore suspension is added into a lithium chloride solution, and the solution is placed under an ultraviolet lamp for irradiation for 30-35cm, the power of the ultraviolet lamp is 30W, and the irradiation time under the ultraviolet lamp is 20-80 s.
Further, in the step (2), the concentration of NTG is 5mg/ml, and the volume ratio of NTG to the volume of germinated spore suspension is 1: 20.
further, in the step (2), the water bath temperature is 28 ℃, and the water bath heat preservation time is 30 min.
Further, in the step (2), the volume of the physiological saline is the same as the volume of the germinated spore suspension.
Further, in the step (3), the plate medium liquid loading amount is the same as the slant medium liquid loading amount.
Further, in the step (3), the volume of the mutagenic bacteria liquid is the same as the volume of NTG.
Further, in the step (3), the plate culture medium is placed in a constant temperature incubator at 28 ℃ for light-shielding culture for 6-9 days.
Further, in the step (3), the re-culture condition is that the culture is carried out for 9 to 12 days in a constant temperature incubator at 28 ℃.
Further, in the step (3), the plate culture medium is prepared from glucose, yeast extract, peptone, beef extract, and FeSO4·7H2O, agar powder, plate-frame wastewater and water, wherein the pH value is 7.0-7.2, and the mixture is sterilized at 121 ℃ for 30 min.
Further, in the step (3), the plate culture medium is prepared from 0.6% of glucose, 0.15% of yeast extract, 0.15% of peptone, 0.1% of beef extract and FeSO in parts by weight4·7H20.001% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water.
Further, in the step (3), the slant culture medium is prepared from glucose, yeast extract, aspartic acid, beef extract and KH2PO4·3H2O, agar powder, plate-frame wastewater and water, wherein the pH value is 7.0-7.2, and the mixture is sterilized at 121 ℃ for 30 min.
Further, in the step (3), the slant culture medium comprises, by weight, 2% of glucose, 0.15% of yeast extract, 0.1% of aspartic acid, 0.2% of beef extract and KH2PO4·3H20.06 percent of O, 2 percent of agar powder, 30 percent of plate-frame wastewater and the balance of water.
Further, in the step (4), the seed shake flask culture medium is cultured for 36 hours at the temperature of 28-28.5 ℃ and the rotating speed of 220 r/min.
Further, in the step (4), the fermentation shake flask culture medium is cultured for 12-13 days at the temperature of 28-28.5 ℃ and the rotating speed of 220 r/min.
Further, in the step (4), strains with the shaking table titer of more than 6000ug/ml, the bacterial concentration of 38-42% and the pH value of 6.3-6.5 are screened out.
Further, in the step (4), the seed shake flask culture medium is composed of corn starch, soybean cake powder, peanut cake powder, yeast powder, amylase and water, the pH value is 7.0-7.2, and the seed shake flask culture medium is sterilized at 121 ℃ for 30 min.
Furthermore, in the step (4), the seed shake flask culture medium comprises, by weight, 3% of corn starch, 2.5% of soybean cake powder, 0.65% of peanut cake powder, 0.3% of yeast powder, 0.0025% of amylase, and the balance of water.
Further, in the step (4), the fermentation shake flask culture medium is prepared from corn starch, soybean cake powder, yeast extract and CoCl2·6H2O, calcium carbonate, ammonium sulfate, manganese sulfate, sodium molybdate, amylase and water, wherein the pH value is 7.0-7.2, and the sterilization is carried out for 30min at 121 ℃.
Further, in the step (4), the fermentation shake flask culture medium is prepared from 14% of corn starch, 2.5% of soybean cake powder, 1.0% of yeast powder, 3.0% of yeast extract and CoCl in parts by weight2·6H20.003 percent of O, 1.0 percent of calcium carbonate, 0.03 percent of ammonium sulfate, 0.0002 percent of manganese sulfate, 0.002 percent of sodium molybdate, 0.0025 percent of amylase, and the balance of plate frame wastewater and water, wherein the plate frame wastewater accounts for 30 percent of the volume after digestion.
Further, in the step (4), the culture conditions of the seed shake flask culture medium are 28-28.5 ℃ and the rotation speed of 220r/min for culturing 36-42h, and the culture conditions of the fermentation shake flask culture medium are 28-28.5 ℃ and the rotation speed of a shaking table of 220r/min for culturing 12-13 days.
Further, after the strain is obtained in the step (4), repeated passage and rejuvenation of the strain are carried out.
The avermectin strain provided by the invention has the average titer of 5800-; meanwhile, 30% of discharge amount of tap water for fermentation and plate frame wastewater is reduced, and the method has the environmental protection significance of protecting water resources; the production and operation cost of enterprises is reduced.
Preservation description:
the preservation unit: china general microbiological culture Collection center,
the preservation unit is abbreviated as: CGMCC (China general microbiological culture Collection center)
Address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing,
the preservation number is: the CGMCC No.17157 is used as the raw material,
the preservation date is as follows: year 2019, month 1 and day 10.
Detailed Description
The invention will be further described with reference to specific embodiments, the advantages and features of which will become apparent from the description, but which are given by way of illustration only and are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
(I) detection of potency
Determination of titer of effective components in fermentation liquor
1 apparatus
A vortex mixer; a centrifuge tube with a plug: 10ml and 100 ml; microsyringe: 100 ul; high performance liquid chromatograph: a variable wavelength ultraviolet detector; a data processing system; a chromatographic column: 150 x 4.6mm (id) stainless steel C18, 5 um; a filter: the aperture of the filter membrane is about 0.45 um; flat head microsyringe: 100 ul; a centrifuge: 3500 rpm.
2 operating conditions of high performance liquid chromatography
Mobile phase: methanol by weight: water 86: 14; the flow rate is 1.0 ml/min; column temperature: room temperature; detection wavelength: 245 nm; sample introduction volume: 60 ul.
3 reagents and solutions
Methanol: carrying out chromatographic purification; water: meets the first-grade water in GB/T6682; the abamectin standard substance: the mass fraction of the abamectin B1 is known to be more than or equal to 98.0 percent, and the mass fraction of B1a is known.
4 operation step
4.1 preparation of samples
4.1.1 defoaming: the fermentation broth was placed in a 100ml centrifuge cup and centrifuged at 3500 rpm for 1 minute in a centrifuge, and then taken out and sufficiently stirred with a glass rod.
4.1.2 preparation: accurately sucking 1 ml of the uniform fermentation liquid after defoaming into a 10ml volumetric flask, adding 5ml of methanol, performing ultrasonic treatment for 15-20min, taking out, standing to room temperature, dissolving and diluting to a scale with methanol, and shaking up. Standing, and filtering the supernatant with 0.45um microporous membrane.
5, preparing a standard sample: accurately weighing 0.04 g of abamectin standard product to be accurate to 0.0002 g, dissolving in a 50 ml volumetric flask, dissolving with methanol, fixing the volume to the scale, and shaking up. Transfer 1 ml to 10ml volumetric flask, dissolve with methanol and fix to the mark.
And 6, under the conditions, after the instrument is stable, injecting a standard sample solution, and after the peak area of the continuous abamectin standard samples is relatively changed by less than 1.5%, injecting the prepared sample solution into a trace sample injector for detection.
7 calculation of
Correction factor F ═ mSign board×P/ASign board
Titer B1a (ug/ml) is the dilution multiple × of a × F × standard
In the formula: m isSign boardMass of standard, g
The content of B1a percent of the P-abamectin standard product
ASign boardPeak area of Standard B1a
A- -area of peak of sample B1a
(II) determination of bacterial concentration
Determination of fermentation liquor bacterial concentration
1 apparatus
Centrifuging the tube: 10 ml; a straw is calibrated: 10 ml; a centrifuge: 3500 r/min.
2 operating procedure
2.1 sucking 10ml of fermentation liquid by using a 10ml graduated pipette, transferring the 10ml of fermentation liquid into a 10ml centrifugal tube, putting the centrifugal tube with the fermentation liquid into a centrifugal machine, setting the rotating speed of the centrifugal machine to be 3500r/min, and centrifuging for 10 min.
And 2.2, taking out the centrifuge tube after the centrifugation time is up, pouring out the supernatant to leave dry matter, and calculating the volume percentage of the dry matter in 10ml of fermentation liquor, namely the microbial concentration.
Example 1
(1) Preparation of germinated spore suspension: take 1cm2Placing the slant in 20ml sterile water to make the spore fully dispersed in the water to obtain spore suspension, placing the triangular flask with the spore suspension on a shaking bed, and culturing at 28 deg.C at 100r/min for 1.5h to obtain germinated spore suspension;
(2) complex mutagenesis of germinated spore suspension: adding 10ml of germinated spore suspension into a lithium chloride solution with the mass concentration of 0.3%, then placing the solution under a UV ultraviolet lamp for irradiating for 20s at a position of 30cm, wherein the power of the ultraviolet lamp is 30W, stirring the solution, then adding 0.5ml of NTG with the concentration of 5mg/ml into a bacterial solution treated by ultraviolet light-lithium chloride, immediately placing the mixture into a water bath with the temperature of 28 ℃ after uniformly mixing, preserving the temperature for 30min, then adding 9ml of normal saline, and shaking the mixture uniformly to obtain a mutagenic bacterial solution;
(3) plate and slant culture: 0.5ml of mutagenic bacteria liquid is sucked and diluted by 105After doubling, coating the diluent on a plate culture medium containing plate-and-frame wastewater, culturing for 6 days at 28 ℃, adding the diluent into a slant culture medium, and culturing for 9 days at 28 ℃ to obtain a seed solution;
(4) shaking table cultivation and screening: inoculating 40ml of seed liquid into a seed shake flask culture medium, and culturing for 36h at 28 ℃ and the rotating speed of 220 r/min. Then transferring the seeds into a fermentation shake flask culture medium for culture, wherein the liquid filling amount is the same as that of a seed shake flask; culturing at 28 deg.C and rotation speed of 220r/min for 12 days, detecting the bacterial concentration, pH value and titer of shake flask fermentation liquid, and screening out strains with shaking table titer of more than 6000ug/ml, bacterial concentration of 38-42%, and pH of 6.3-6.5;
(5) rejuvenation of the strains: repeatedly carrying out passage rejuvenation on the screened strains to ensure that the heredity of the strains is stable;
(6) tank casting production: and (3) carrying out large-tank production and use on the mutagenic high-titer strains verified by a shaking table.
Wherein the content of the first and second substances,
the plate culture medium comprises, by weight, 0.6% of glucose, 0.15% of yeast extract, 0.15% of peptone, 0.1% of beef extract, and FeSO4·7H20.001% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30 min;
the slant culture medium comprises 2% of glucose, 0.15% of yeast extract, 0.1% of aspartic acid, 0.2% of beef extract and KH2PO4·3H20.06% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30min
The seed shake flask culture medium comprises, by weight, 3% of corn starch, 2.5% of soybean cake powder, 0.65% of peanut cake powder, 0.3% of yeast powder, 0.0025% of amylase and the balance of water, wherein the pH value is 7.2, and the seed shake flask culture medium is sterilized at 121 ℃ for 30 min.
The fermentation shake flask culture medium comprises 14 weight parts of corn starch, 2.5 weight parts of soybean cake powder, 1.0 weight part of yeast powder, 3.0 weight parts of yeast extract and CoCl2·6H20.003% of O, 1.0% of calcium carbonate, 0.03% of ammonium sulfate, 0.0002% of manganese sulfate, 0.002% of sodium molybdate, 0.0025% of amylase, and the balance of plate and frame wastewater and water, wherein the pH value is 7.2, the plate and frame wastewater accounts for 30% of the volume after disinfection, and the sterilization is carried out for 30min at 121 ℃.
Example 2
(1) Preparation of germinated spore suspension: take 1cm2Placing the slant in 20ml sterile water to make the spore fully dispersed in the water to obtain spore suspension, placing the triangular flask with the spore suspension on a shaking bed, and culturing at 28.5 deg.C at 100r/min for 2h to obtain germinated spore suspension;
(2) complex mutagenesis of germinated spore suspension: adding 10ml of germinated spore suspension into a lithium chloride solution with the mass concentration of 0.5%, then placing the solution under a UV ultraviolet lamp for irradiating for 80s at a position of 35cm, wherein the power of the ultraviolet lamp is 30W, stirring the solution, then adding 0.5ml of NTG with the concentration of 5mg/ml into a bacterium solution treated by ultraviolet light-lithium chloride, immediately placing the mixture into a water bath with the temperature of 28.5 ℃ after uniformly mixing the mixture for heat preservation for 30min, then adding 9ml of normal saline, and shaking the mixture uniformly to obtain a mutagenic bacterium solution;
(3) plate and slant culture: 0.5ml of compound mutagenized bacteria liquid is sucked and diluted by 10 percent6After doubling, coating the diluent on a plate culture medium containing plate-and-frame wastewater, culturing for 6 days at 28.5 ℃, then adding the diluent into a slant culture medium, and culturing for 9 days at 28.5 ℃ to obtain a seed solution;
(4) shaking table cultivation and screening: inoculating 40ml of seed liquid into a seed shake flask culture medium, and culturing for 42h at 28.5 ℃ and the rotation speed of 220 r/min. Then transferring the strain into a fermentation shake flask culture medium for culture, wherein the liquid loading amount is the same as that of a seed shake flask culture medium; culturing at 28.5 deg.C and rotation speed of 220r/min for 13 days, detecting the bacterial concentration, pH value and titer of shake flask fermentation liquid, and screening out strains with shaking table titer of more than 6000ug/ml, bacterial concentration of 38-42%, and pH of 6.3-6.5;
(5) rejuvenation of the strains: repeatedly carrying out passage rejuvenation on the screened strains to ensure that the heredity of the strains is stable;
(6) tank casting production: and (3) carrying out large-tank production and use on the mutagenic high-titer strains verified by a shaking table.
Wherein the content of the first and second substances,
the plate culture medium comprises, by weight, 0.6% of glucose, 0.15% of yeast extract, 0.15% of peptone, 0.1% of beef extract, and FeSO4·7H20.001% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.0; sterilizing at 121 deg.C for 30 min;
the slant culture medium comprises 2% of glucose, 0.15% of yeast extract, 0.1% of aspartic acid, 0.2% of beef extract and KH2PO4·3H20.06% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.0; sterilizing at 121 deg.C for 30 min;
the seed shake flask culture medium comprises, by weight, 3% of corn starch, 2.5% of soybean cake powder, 0.65% of peanut cake powder, 0.3% of yeast powder, 0.0025% of amylase and the balance of water, wherein the pH value is 7.0, and the seed shake flask culture medium is sterilized at 121 ℃ for 30 min.
The fermentation shake flask culture medium comprises 14 weight parts of corn starch, 2.5 weight parts of soybean cake powder, 1.0 weight part of yeast powder, 3.0 weight parts of yeast extract and CoCl2·6H20.003% of O, 1.0% of calcium carbonate, 0.03% of ammonium sulfate, 0.0002% of manganese sulfate, 0.002% of sodium molybdate, 0.0025% of amylase, and the balance of plate and frame wastewater and water, wherein the pH value is 7.0, the plate and frame wastewater accounts for 30% of the volume after disinfection, and the sterilization is carried out for 30min at 121 ℃.
Comparative example 1 in comparison with example 1, the composition of the plate medium and the slant medium was different
(1) Preparation of germinated spore suspension: take 1cm2Placing the slant in 20ml sterile water to make the spore fully dispersed in the water to obtain spore suspension, placing the triangular flask with the spore suspension on a shaking bed, and culturing at 28 deg.C at 100r/min for 1.5h to obtain germinated spore suspension;
(2) complex mutagenesis of germinated spore suspension: adding 10ml of germinated spore suspension into a lithium chloride solution with the mass concentration of 0.3%, then placing the solution under a UV ultraviolet lamp for irradiating for 20s at a position of 30cm, wherein the power of the ultraviolet lamp is 30W, stirring the solution, then adding 0.5ml of NTG with the concentration of 5mg/ml into a bacterial solution treated by ultraviolet light-lithium chloride, immediately placing the solution in a water bath with the temperature of 28 ℃ after uniformly mixing the solution and preserving the heat for 30min, and then adding 9ml of physiological saline and shaking the solution uniformly to obtain a mutagenic bacterial solution;
(3) plate and slant culture: 0.5ml of compound mutagenized bacteria liquid is sucked and diluted by 10 percent5After doubling, coating the diluent on a plate culture medium containing plate-and-frame wastewater, culturing for 6 days at 28 ℃, adding the diluent into a slant culture medium, and culturing for 9 days at 28 ℃ to obtain a seed solution;
(4) shaking table cultivation and screening: inoculating 40ml of seed liquid into a seed shake flask culture medium, and culturing for 36h at 28 ℃ and the rotating speed of 220 r/min. Then transferring the strain into a fermentation shake flask culture medium for culture, wherein the liquid loading amount is the same as that of a seed shake flask culture medium; culturing at 28 deg.C and rotation speed of 220r/min for 12 days, detecting the bacterial concentration, pH value and titer of shake flask fermentation liquid, and screening out strains with shaking table titer of more than 6000ug/ml, bacterial concentration of 38-42%, and pH of 6.3-6.5;
(5) rejuvenation of the strains: repeatedly carrying out passage rejuvenation on the screened strains to ensure that the heredity of the strains is stable;
(6) tank casting production: and (3) carrying out large-tank production and use on the mutagenic high-titer strains verified by a shaking table.
Wherein the content of the first and second substances,
the plate culture medium consists of 0.6 percent of glucose, 0.15 percent of ammonium sulfate, 0.8 percent of sodium carbonate, 2 percent of agar powder, 30 percent of plate frame wastewater and the balance of water in parts by weight, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30 min;
the slant culture medium comprises, by weight, 0.6% of glucose, 0.15% of ammonium sulfate, 0.8% of sodium carbonate, 2% of agar powder, 30% of plate-and-frame wastewater and the balance of water, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30 min;
the seed shake flask culture medium comprises 3% of corn starch, 2.5% of soybean cake powder and CoCl2·6H2O0.003%, amylase 0.0025%, pH 7.2, prepared with tap water, and sterilized at 121 deg.C for 30 min.
The fermentation shake flask culture medium is prepared from 3% of corn starch, 2.5% of peanut cake powder and CoCl2·6H20.003% of O, 0.0025% of amylase, plate-frame wastewater and the balance of water, wherein the pH value is 7.2, the plate-frame wastewater accounts for 30% of the volume after digestion, and the plate-frame wastewater is sterilized at 121 ℃ for 30 min.
Comparative example 2 the preparation method differs from example 1 in step (2)
(1) Preparation of germinated spore suspension: take 1cm2Placing the slant in 20ml sterile water to make the spore fully dispersed in the water to obtain spore suspension, placing the triangular flask with the spore suspension on a shaking bed, and culturing at 28 deg.C at 100r/min for 1.5h to obtain germinated spore suspension;
(2) complex mutagenesis of germinated spore suspension: adding 10ml of germinated spore suspension into a lithium chloride solution with the mass concentration of 1%, then placing the solution under a UV ultraviolet lamp for 30cm to irradiate for 20s, wherein the power of the ultraviolet lamp is 30W, stirring the solution, then adding 0.5ml of NTG with the concentration of 10mg/ml into the bacterium solution treated by the ultraviolet light-lithium chloride, immediately placing the mixture into a water bath with the temperature of 30 ℃ after uniformly mixing, preserving the heat for 30min, then adding 9ml of normal saline, and shaking the mixture uniformly to obtain a mutagenic bacterium solution;
(3) plate and slant culture: 0.5ml of compound mutagenized bacteria liquid is sucked and diluted by 10 percent5After doubling, coating the diluent on a plate culture medium containing plate-and-frame wastewater, culturing for 6 days at 28 ℃, adding the diluent into a slant culture medium, and culturing for 9 days at 28 ℃ to obtain a seed solution;
(4) shaking table cultivation and screening: inoculating 40ml of seed liquid into a seed shake flask culture medium, and culturing for 36h at 28 ℃ and the rotating speed of 220 r/min. Then transferring the strain into a fermentation shake flask culture medium for culture, wherein the liquid loading amount is the same as that of a seed shake flask culture medium; culturing at 28 deg.C and rotation speed of 220r/min for 12 days, detecting the bacterial concentration, pH value and titer of shake flask fermentation liquid, and screening out strains with shaking table titer of more than 6000ug/ml, bacterial concentration of 38-42%, and pH of 6.3-6.5;
(5) rejuvenation of the strains: repeatedly carrying out passage rejuvenation on the screened strains to ensure that the heredity of the strains is stable;
(6) tank casting production: and (3) carrying out large-tank production and use on the mutagenic high-titer strains verified by a shaking table.
Wherein the content of the first and second substances,
the plate culture medium comprises, by weight, 0.6% of glucose, 0.15% of yeast extract, 0.15% of peptone, 0.1% of beef extract, and FeSO4·7H20.001% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30 min;
the slant culture medium comprises 2% of glucose, 0.15% of yeast extract, 0.1% of aspartic acid, 0.2% of beef extract and KH2PO4·3H20.06% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30 min;
the seed shake flask culture medium comprises, by weight, 3% of corn starch, 2.5% of soybean cake powder, 0.65% of peanut cake powder, 0.3% of yeast powder, 0.0025% of amylase and the balance of water, wherein the pH value is 7.2, and the seed shake flask culture medium is sterilized at 121 ℃ for 30 min. The fermentation shake flask culture medium comprises 14 weight parts of corn starch, 2.5 weight parts of soybean cake powder, 1.0 weight part of yeast powder, 3.0 weight parts of yeast extract and CoCl2·6H20.003 percent of O, 1.0 percent of calcium carbonate, 0.03 percent of ammonium sulfate, 0.0002 percent of manganese sulfate, 0.002 percent of sodium molybdate,0.0025% of amylase, plate and frame wastewater and the balance of water, wherein the pH is 7.2, the plate and frame wastewater accounts for 30% of the volume after digestion, and the sterilization is carried out for 30min at 121 ℃.
Comparative example 3 the preparation process differs from example 1 in the steps (3), (4)
(1) Preparation of germinated spore suspension: take 1cm2Placing the slant in 20ml sterile water to make the spore fully dispersed in the water to obtain spore suspension, placing the triangular flask with the spore suspension on a shaking bed, and culturing at 28 deg.C at 100r/min for 1.5h to obtain germinated spore suspension;
(2) complex mutagenesis of germinated spore suspension: adding 10ml of germinated spore suspension into a lithium chloride solution with the mass concentration of 0.3%, then placing the solution under a UV ultraviolet lamp for irradiating for 20s at a position of 30cm, wherein the power of the ultraviolet lamp is 30W, stirring the solution, then adding 0.5ml of NTG with the concentration of 5mg/ml into a bacterial solution treated by ultraviolet light-lithium chloride, immediately placing the solution in a water bath with the temperature of 28 ℃ after uniformly mixing the solution and preserving the heat for 30min, and then adding 9ml of physiological saline and shaking the solution uniformly to obtain a mutagenic bacterial solution;
(3) plate and slant culture: 0.5ml of compound mutagenized bacteria liquid is sucked and diluted by 10 percent5After doubling, coating the diluent on a plate culture medium containing plate-and-frame wastewater, culturing for 2 days at 30 ℃, adding the diluent into a slant culture medium, and culturing for 5 days at 25 ℃ to obtain a seed solution;
(4) shaking table cultivation and screening: inoculating 40ml of seed liquid into a seed shake flask culture medium, and culturing for 10h at 35 ℃ and the rotating speed of 220 r/min. Then transferring the strain into a fermentation shake flask culture medium for culture, wherein the liquid loading amount is the same as that of a seed shake flask culture medium; culturing at 20 deg.C and rotation speed of 220r/min for 20 days, detecting the bacterial concentration, pH value and titer of shake flask fermentation liquid, and screening out strains with shaking table titer of more than 6000ug/ml, bacterial concentration of 38-42%, and pH of 6.3-6.5;
(5) rejuvenation of the strains: repeatedly carrying out passage rejuvenation on the screened strains to ensure that the heredity of the strains is stable;
(6) tank casting production: and (3) carrying out large-tank production and use on the mutagenic high-titer strains verified by a shaking table.
Wherein the content of the first and second substances,
according to the weightThe plate culture medium comprises, by weight, 0.6% of glucose, 0.15% of yeast extract, 0.15% of peptone, 0.1% of beef extract, and FeSO4·7H20.001% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30 min;
the slant culture medium comprises 2% of glucose, 0.15% of yeast extract, 0.1% of aspartic acid, 0.2% of beef extract and KH2PO4·3H20.06% of O, 2% of agar powder, 30% of plate-frame wastewater and the balance of water, wherein the pH value is 7.2; sterilizing at 121 deg.C for 30 min;
the seed shake flask culture medium comprises, by weight, 3% of corn starch, 2.5% of soybean cake powder, 0.65% of peanut cake powder, 0.3% of yeast powder, 0.0025% of amylase and the balance of water, wherein the pH value is 7.2, and the seed shake flask culture medium is sterilized at 121 ℃ for 30 min.
The fermentation shake flask culture medium comprises 14 weight parts of corn starch, 2.5 weight parts of soybean cake powder, 1.0 weight part of yeast powder, 3.0 weight parts of yeast extract and CoCl2·6H20.003% of O, 1.0% of calcium carbonate, 0.03% of ammonium sulfate, 0.0002% of manganese sulfate, 0.002% of sodium molybdate, 0.0025% of amylase, and the balance of plate and frame wastewater and water, wherein the pH value is 7.2, the plate and frame wastewater accounts for 30% of the volume after disinfection, and the sterilization is carried out for 30min at 121 ℃.
EXAMPLE 1 potency assay
According to the above-mentioned titer determination method, the titer of the product B1a obtained after one week of culture of the strains cultured according to the methods of examples 1-2 and comparative examples 1-3 was determined, and the experimental results were as follows:
| group of | Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Potency of the drug | 6852 | 6421 | 4163 | 4620 | 3849 |
Experimental example 2 COD test of wastewater
The COD of the waste water after the culture is measured by adopting a water quality analyzer, and the experimental result is as follows:
the experimental results show that the strain can be suitable after mutation domestication, and residual nutrient substances and organic acid in wastewater can be utilized without influencing the tank placing titer of a fermentation tank; meanwhile, 30% of discharge amount of tap water for fermentation and plate frame wastewater is reduced, the COD value in water is small, and the method has the environmental protection significance of protecting water resources; reduces the production and operation cost of enterprises
The technical means disclosed by the scheme of the invention are not limited to the technical means disclosed above, and the technical means also comprises the technical scheme formed by any combination of the technical features. While the foregoing is directed to embodiments of the present invention, it is noted that various changes and modifications may be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (1)
1. The avermectin strain is characterized by being named as Streptomyces avermitilis by classification, the Latin by classification is named as Streptomyces avermitilis, the preservation unit is China general microbiological culture Collection center, the preservation number is CGMCC No.17157, and the preservation date is 2019, 1 month and 10 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910160954.1A CN109868239B (en) | 2019-03-04 | 2019-03-04 | Abamectin strain and screening method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910160954.1A CN109868239B (en) | 2019-03-04 | 2019-03-04 | Abamectin strain and screening method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109868239A CN109868239A (en) | 2019-06-11 |
| CN109868239B true CN109868239B (en) | 2020-06-30 |
Family
ID=66919690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910160954.1A Active CN109868239B (en) | 2019-03-04 | 2019-03-04 | Abamectin strain and screening method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109868239B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305818A (en) * | 2019-08-15 | 2019-10-08 | 齐鲁制药（内蒙古）有限公司 | A kind of avermectin strain breeding method |
| CN111979283B (en) * | 2020-08-13 | 2021-05-28 | 内蒙古新威远生物化工有限公司 | Method for producing abamectin by fermenting composite strains |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154168B (en) * | 2011-01-07 | 2012-06-06 | 石家庄市兴柏生物工程有限公司 | Abamectin producing bacterium and preparation method thereof |
| CN104498400A (en) * | 2014-12-10 | 2015-04-08 | 常熟理工学院 | Mutant strain of high-yield abamectin B component and screening method thereof |
| CN106190928B (en) * | 2016-08-31 | 2019-10-29 | 华北制药集团爱诺有限公司 | A kind of avermectin superior strain and its screening technique |
-
2019
- 2019-03-04 CN CN201910160954.1A patent/CN109868239B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109868239A (en) | 2019-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102634471B (en) | Avermectin B1a high-yielding strain and application thereof | |
| CN109868239B (en) | Abamectin strain and screening method thereof | |
| CN113980833B (en) | Bacillus megaterium and application thereof in soil phosphate solubilizing | |
| CN104263682B (en) | Plant-growth-promoting endophytic bacterium having polycyclic aromatic hydrocarbons degrading function and application thereof | |
| CN111676156A (en) | Bacillus belgii MRS for improving reduction activity and fermentation product and application thereof | |
| CN107446842B (en) | Bacillus subtilis and application thereof in water purification | |
| CN103740606A (en) | Streptomyces phytohabitans, method for producing new antibiotics Novonestmycin from Streptomyces phytohabitans, and application of Novonestmycin | |
| CN102776137B (en) | Application of microorganism bacterium agent in biological treatment of phenolic wastewater | |
| CN101096646A (en) | Ferrum reduction bacterium-mineral composite agent capable of accelerating organic chlorine degradation and preparation method thereof | |
| CN109576171B (en) | Lysinibacillus fusiformis and application thereof | |
| CN105754890A (en) | Streptomyces hygroscopicus for producing validamycin and application of streptomyces hygroscopicus | |
| CN108467879B (en) | Synthetic medium for erythromycin fermentation | |
| CN116904354A (en) | Priestia aryabhattai H2 and application thereof | |
| CN111057669A (en) | Strain for improving yield of tetramycin Z and method for preparing tetramycin Z by using same | |
| CN113881602B (en) | High-yield C 21 Steroid bacillus cereus X-32 and application thereof | |
| CN105505798A (en) | Endophytic fungus for generating ergosterol and application of endophytic fungus | |
| CN113817614B (en) | High-efficiency synthesis C 21 Alternaria alternata Z-44 of steroid glycoside and application thereof | |
| CN111154679B (en) | Efficient fermentation method of aflatoxin degradation bacteria | |
| CN111748489B (en) | Marine streptomyces griseofulensis HN60 and application thereof | |
| CN114196583A (en) | Thermophilic carbon monoxide chain mould, synergistic biological agent containing same and application | |
| CN105296379A (en) | Pseudonocardia sp. and application thereof | |
| CN114134079B (en) | Tetracycline antibiotic degrading bacteria, method and application | |
| CN109868247B (en) | Streptomyces TJ138, product, preparation method and application thereof, method for reducing cadmium content in crops and application of melatonin | |
| CN110261267B (en) | Detection method of photosynthetic bacteria preparation product for fishing | |
| CN113025519B (en) | Aeromonas intermedia and application thereof in removing chloramphenicol and dissolving phosphorus and potassium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191023 Address after: 518000 Guangdong city of Shenzhen province Qianhai Shenzhen Hong Kong cooperation zone before Bay Road No. 1 building 201 room A Applicant after: Yang Junqiang Address before: 014300 New Austrian Industrial Park, Dalat Banner, Ordos City, Inner Mongolia Autonomous Region Applicant before: INNER MONGOLIA XINWEIYUAN BIOCHEMICAL CO., LTD. |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200205 Address after: 014300 Xinao Industrial Park, Dalate County, Ordos City, Inner Mongolia Autonomous Region Applicant after: INNER MONGOLIA XINWEIYUAN BIOCHEMICAL CO., LTD. Address before: 518000 Guangdong city of Shenzhen province Qianhai Shenzhen Hong Kong cooperation zone before Bay Road No. 1 building 201 room A Applicant before: Yang Junqiang |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 014300 New Austrian Industrial Park, Dalat Banner, Ordos City, Inner Mongolia Autonomous Region Patentee after: INNER MONGOLIA XINWEIYUAN BIOCHEMICAL Co.,Ltd. Address before: 014300 Xinao Industrial Park, Dalate County, Ordos City, Inner Mongolia Autonomous Region Patentee before: INNER MONGOLIA XINWEIYUAN BIOCHEMICAL Co.,Ltd. |