CN101768586A - Breeding process of high-titer strains of streptomyces avermitilis strains subjected to heavy ion irradiation mutagenesis - Google Patents
Breeding process of high-titer strains of streptomyces avermitilis strains subjected to heavy ion irradiation mutagenesis Download PDFInfo
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- CN101768586A CN101768586A CN 200810187967 CN200810187967A CN101768586A CN 101768586 A CN101768586 A CN 101768586A CN 200810187967 CN200810187967 CN 200810187967 CN 200810187967 A CN200810187967 A CN 200810187967A CN 101768586 A CN101768586 A CN 101768586A
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Abstract
The invention relates to a breeding process of high-titer strains of streptomyces avermitilis strains subjected to heavy ion irradiation mutagenesis, belonging to the field of antibiotic breeding. The breeding process further researches breeding to obtain an optimal breeding process after heavy ion irradiation and is characterized in that fermentation temperature is (28+/-0.6) DEG C; the pH of a fermentation medium is 6.5-7.5; the rotation speed of a bottle rocker is 220-260 r/min; the inoculation age is 22-26 hours; the inoculation amount is 4-5 percent; and the fermentation medium comprises the following components: starch, dextrin, bean cake powder, peanut cake powder, yeast powder, yeast cream, cobalt chloride, bean oil and water. The invention has high fermentation titer of more than 6000 microgrammes/milliliter, reliable, controllable, adjustable and stable process, rich culture medium resources and low cost.
Description
Technical field
The present invention relates to microbiotic seed selection field, especially the seed selection of A Foman streptomyces strain.
Background technology
Avrmectin be the A Foman streptomycete behind liquid fermenting, the meta-bolites of extraction is ten hexa-atomic macrolide antibiotics.Its preparation can be used as agricultural chemicals, veterinary drug, characteristics such as have wide spectrum, efficient, low toxicity, noresidue, be difficult for developing immunity to drugs.
Aspect agricultural chemicals, Avrmectin is a kind of agricultural antibiotic insecticides, miticide, belongs to the insect nerve poison, mainly disturbs the activity of insect nervous physiology, and its paralysis is poisoned and death.Absorption in not having, but stronger osmosis is arranged, and can be in plant materials cross conduction, desinsection, acaricidal activity height, higher 5～50 times than common pesticides, dosage only is 1%～2% of a common pesticides.This medicament antagonism property of medicine insect has preventive effect preferably, does not have the transreactance of intersection with organophosphorus, pyrethroid and carbamate chemicals for agriculture, and is safer to natural enemy.
Avrmectin is one of Biocidal miticide current of greatest concern, by the Pesticidal products of the Ministry of Agriculture as the high-efficiency low-toxicity of recommending to use.The biological farming of antibiotics, veterinary drug that Avrmectin is processed through liquid fermenting by the A Foman Streptomycin sulphate.Compare with traditional farming, veterinary drug, have efficient, wide spectrum, validity period is long, be difficult for developing immunity to drugs, easily degraded, characteristics such as noresidue, and be difficult for the generation resistance, there is not crossed resistance with other pesticides.Fast in degraded in soil, photodissociation is rapid, to crop safety, is difficult for producing poisoning.Avrmectin is the 21st a big situation of selling well agricultural chemicals in the world, the 4th big situation of selling well sterilant, and sales volume is up to 1,600,000,000 dollars.Because Avrmectin is a kind of new antibiotic class, have novel structure, the dual-purpose characteristics of farming animals, therefore, along with the raising of people's living standard and to the call of green food, this class biological pesticide enjoys favor in current agricultural chemicals market, and Avrmectin also becomes most popular and the most competitive product innovation in the current biological pesticide market.
Contain 8 components in the natural Avrmectin, mainly containing 4 kinds is A1a, A2a, B1a and B2a, its total content 〉=80%; 4 corresponding less homologues of ratio are A1b, A2b, B1b and B2b, its total content≤20%.Present commercially available Avrmectin agricultural chemicals is to be main insecticidal constituent (AvermectinB1a+B1b, wherein B1a is not less than 90%, B1b is no more than 5%) with abamectin, demarcates with the content of B1a.
AVMB1a molecular formula: C
48H
72O
14(R=C
2H
5)
AVMB1a molecular weight: 872
2.3 the physico-chemical property of Avrmectin
Avrmectin is white or faint yellow crystallisate, fusing point is 157-162 ℃, α (CHCl3)=+ 55.7 237,245, all there is absorption peak at the 254nm place, λ max=244 ± 2nm, Avrmectin is dissolved in toluene, ethyl acetate, acetone, trichloromethane, ethanol equal solvent, be slightly soluble in normal hexane and sherwood oil, the solubleness in water extremely low (10 μ g/l), this product should be preserved under dry, airtight, shady and cool shading.No acidic or basic functionality in this product molecule, under general condition stable, can hydrolysis when PH5-9, non-corrosiveness, environmentally safe.
The principal element that influences Chinese microbiotic industry development has: strain fermentation is tired and is promoted slowly, even all can not get breakthrough progress for a long time.
At present, international and domestic microbiotic bacterial classification mutagenesis means are seen the mutagenesis of chemomorphosis method and ordinary beam, low energy ion more, but all do not obtain the ideal fermentation titer, abamectin fermented tiring only at 4000 μ g/ml.Along with the development of China Aerospace cause, the means of space breeding have appearred in recent years.But the limited difficult realization of condition.This project adopts the physical mutagenesis method of heavy ion beam irradiation, have controlled, adjustable, can repeated characteristics, significantly improved the randomness and the uncontrollability of other irradiation means.
During adopting, we can carry out radiation treatment to the Avrmectin bacterial strain by heavy ion beam, for finding out the optimum parameter of suitable Avrmectin bacterial strain mutagenesis, the calculating of irradiation physical parameter has been carried out repeatedly optimizing and revising, handled, determined best irradiation dose and medial lethal dose the Avrmectin bacterial classification through 4 irradiation.Then after testing laboratory carries out repeated screening, obtain that production performance is good, the bacterial strain of good stability, by optimizing fermention medium, its fermentation titer has been reached more than the 5800 μ g/ml again.These technological achievements are recorded in " the heavy ion irradiation mutagenesis of A Foman streptomyces strain and irradiation spawn culture method ", and have carried out patent application.
Microbial fermentation generally is divided into fermentation and extracts two stages, comprises that spore preparation, slant culture, shake-flask culture, seed flask culture, fermentation flask fermentation and mash extract.In the whole process flow of microbial fermentation, one step of most critical is exactly a fermentation stage, fermentation is subjected to the influence of many factors, we must keep a close eye on just and can achieve the desired result, the principal element of some influence fermentations has the pollution of assorted bacterium and phage, add nutraceutical supplying technics, best omnidistance effective monitoring of producing anti-pH, the omnidistance effective monitoring of temperature, ventilation, stirring and foam control etc., the bacterial classification that the key condition of these decision fermentation success or failure obtains different mutagenesis is different, as a bacterial classification that obtains by heavy ion irradiation mutagenesis its exclusive processing condition are arranged also, therefore we must do further to explore on the basis of leading portion test, so that obtain best breeding process condition.
Summary of the invention
Purpose of the present invention is for providing the A Foman streptomyces strain through heavy ion
12C
+ 6Height behind the irradiation mutagenesis strain improvement technology of tiring is by this breeding process, to obtain good quality strain and to reduce production costs.
The processing condition that realize the foregoing invention purpose are:
The anti-processing condition of product through the gain mutant bacterial strain of heavy ion irradiation mutagenesis A Foman streptomyces strain are: shaking table machine rotating speed 220-260r/min, inoculum size 4% that seed culture medium PH 7.0-7.2, leavening temperature be 28 ± 0.6 ℃, fermention medium PH 6.5-7.5, regulate oxygen solubility.
Content of the test
Bacterial classification S.avermitilis ZJAV-y II sets out
Warp
12C
+ 6Heavy ion irradiation
Irradiation dose: 50-70Gy
Test subject comprises: the shaking table machine stirring velocity of age, pH value, inoculum size and control dissolved oxygen is planted in slant culture, shake flask fermentation, mensuration leavening temperature, inoculation.
1. temperature
In the leavening temperature scope, along with the rising of temperature, thalli growth and metabolism are accelerated, and the speed of fermentation reaction is accelerated.After surpassing optimum temperature range, along with the rising of temperature, the very fast inactivation of enzyme, the thalline aging, fermentation period shortens, and output reduces.Mainly investigate the fermentation of 24 ℃, 26 ℃, 28 ℃ three temperature condition, followed the tracks of the difference that has compared the variation of nutraceutical consumption, pH and the amplification of tiring.
Obviously unusual 24 ℃ of bottom fermentation metabolism, the sugar consumption is on the low side, and mycelial growth rate is slow excessively, and pH remains on high level always, does not resemble the very strong downtrending of appearance under other two temperature, and this reflects that also 24 ℃ of metabolism are inactive.In addition, under 24 ℃, amino nitrogen is higher always, and this also influences thalli growth.Bacterial metabolism is greatly unusual, causes tiring significantly reducing, and finds out that obviously low temperature is bigger to the negative impact of fermentation.
Under 26 ℃, the beginning of sugar consumption is faster than 28 ℃, but mycelial growth difference and not obvious mainly is because the flow of elementary metabolic each bar approach changes.In early stage, the amplification difference of tiring under 28 ℃ and 26 ℃ is also not obvious, and instruction book is from the biosynthesizing of Avrmectin, and two temperatures does not produce significant difference; But to the later stage, the amplification of tiring under 26 ℃ is big not as 28 ℃, and the sugar level of two temperature fermentations is suitable, therefore, two anti-differences of product of following later stage of temperature may be relevant with the quality of thalline, so preceding sugar futures consumption is unsuitable too fast, otherwise can cause thalline to produce anti-time length shortening.
In addition, cenobium is tightr in the time of 28 ℃, and that 26 ℃ of following cenobiums show slightly is loose, because loose cenobium makes the too high and then influence of fermentation broth viscosity produce anti-in jar.Therefore, test-results is the optimum temps section for 28 ℃ ± 0.6 ℃.
2. dissolved oxygen
The supply of oxygen is a key factor concerning aerobic fermentation.Bacterial classification can only utilize the dissolved oxygen in the fermented liquid during the fermentation, therefore, must be in fermented liquid a large amount of oxygen of continuous supplementation, and to constantly stir, can improve the solubleness of oxygen in fermented liquid like this.In the abamectin fermenting process, dissolved oxygen often becomes limiting factor.The oxygen supply deficiency causes the bad and microbiotic output decline of thalli growth, but its mycelia is very sensitive to shearing.Therefore in fermentation, be easy to mycelia and disperse, rupture, cause viscosity too high if control is improper, in case this abnormal phenomenon occurs, can not obtain very high tiring.In general, the early stage of fermentation, mycelia was also more tender and lovely because bacterium shape prepattern still, sent out easily when being subjected to intensive and shearing and stuck with paste; We can say that shearing is to send out the immediate cause of sticking with paste.
Carry out brute force stirring (220-260r/min) by adjusting the rotary shaker machine, improve the solubleness of oxygen in fermented liquid and promptly increase dissolved oxygen.Show through the test of many times result: in abamectin fermented bottle was cultivated in 10-11 days, the shaking table that can not stop was constantly observed fermentation flask color, viscosity, smell.The shaking table that stops can reduce the fermentation flask dissolved oxygen amount, influences the content of Avrmectin B1a, reduces the anti-ability of product of Avrmectin.The suitableeest rotating speed is 250r/min.
3. transferred species kind age
The access seed shakes the seed in the bottle, and along with the prolongation of incubation time, the mycelia amount increases, but owing to the consumption of matrix, the accumulation and the mycelial death of meta-bolites, the no longer increase of mycelia amount, and be tending towards aging gradually.Therefore selecting changes very important that the suitable inoculation of fermentation flask seems age by the seed bottle.Select the mycelium in inoculation age to be in the very vigorous logarithmic phase of vitality, and biomass is inoculated and is the best also when reaching the climax in the nutrient solution, kind changed to 26 hours from 24 hours age, and PH sharply descends; Selected 24 hours to be that the seed bottle changes the fermentation flask Best Times.Plant under the situation of age from 22 hours to 24 hours low spore inoculating amounts, PH slowly rises, and the PH decrement phase is delayed, and under the high spore inoculating amount, pH value obviously descends, and illustrates under the high spore inoculating amount, and the PH decrement phase in advance.
Low spore amount is inoculated under the situation of seed bottle on the inclined-plane, and selecting 26 hours is that the seed bottle changes the fermentation flask Best Times.High spore amount is inoculated under the situation of seed bottle on the inclined-plane, and selecting 22 hours is that the seed bottle changes the fermentation flask Best Times.
4. inoculum size
The size of inoculum size depends on the speed of producing bacterial classification growth and breeding in fermentation shake flask.Adopt bigger inoculum size can shorten the time on mycelia breeding arrival peak in the fermentation shake flask, the formation of product is arrived in advance.This is because grain weight is many, contains a large amount of extracorporeal hydrolysis enzymes in the seed liquor simultaneously, help the utilization of matrix and synthesizing of product, and the production rhzomorph has occupied the chance that whole culture environment has reduced varied bacteria growing rapidly.But, if inoculum size is too much, often make mycelial growth too fast, the nutrient solution viscosity increases, and causes the dissolved oxygen deficiency, and influences the synthetic of product.And inoculum size is too small, except prolonging fermentation period, causes other abnormal conditions toward the contact meeting, sometimes a large amount of hypha bodies can occur.Therefore to inoculum size be controlled at about 4-5% to best in the fermented liquid ratio.
5.pH
In the microbial growth process, must keep certain pH value, but fermenting process often pH might influence microorganism to the absorption of nutritive substance and the secretion of meta-bolites.In addition, pH also can influence the decomposition of nutritive substance in the substratum etc.Therefore, should control the pH of fermented liquid, test-results shows pH 6.0-7.5 the best, and is too high or too low, all can lower the ability of fermentation.Inclined-plane PH transfers to 7.5 ± 0.1, and seed culture medium PH transfers to 7.2 ± 0.1, and fermention medium PH transfers to 7.5 ± 0.1 for good.
In sum, during the fermentation, strict controlled temperature makes its temperature in 28 ℃ ± 0.6 ℃ scope; Transfer pH in the 6.0-7.5 scope; Will regulate the solubleness of oxygen in fermented liquid with the rotating speed of 220-260r/min when cultivating between constant temperature is the best with 250r/min; Inoculum size is at 4-5%.Strictness control and optimization technology by these fermentation conditions give full expression to the throughput of bacterial classification.
AVMB
1aThe test of high density accumulation metabolic regulation
Microorganism can coordinate and balance intravital pathways metabolism of machine and metabolic type mutually by self-control under the normal growth condition, usually can the excess accumulation primary metabolite.But under the control of artificial condition, can make various primary metabolites of the excessive generation of microorganism and secondary metabolite.
Various nutritive substances are synthetic all very important to microorganism growth metabolism and product, but when providing nutritive substance for microorganism, must strictly grasp various nutrient concentrations and ratio, this is not only in order to keep normal osmotic pressure, or save material, be that also nutrient concentrations and ratio directly influence the breeding of thalline and the accumulation of product.
The required nutrition of microorganism growth mainly is carbon source and nitrogenous source two big classes, and they obtain from fermention medium.The carbon source of A Foman streptomycete fermentation substratum is generally starchy material; Organic nitrogen source is groundnut meal, soybean cake powder, corn steep liquor and peptone etc. mostly.Because the fermention medium loading is very big, consume a large amount of grains, increase production cost, might will seek surrogate.In order to prolong the microbial growth phase, increase the output of product, we in conjunction with the crop resources advantage in Gansu, have finally preferably determined suitable Avrmectin high density cumulative carbon source and nitrogenous source on forefathers' basis.
1. the prescription of nutritive substance
The concentration of various nutrient concentrations, particularly carbon-nitrogen ratio, inorganic salt and VITAMIN in the fermented liquid can directly influence the growth of thalline and the accumulation of meta-bolites.
Metabolism curve and the anti-curve of product to the positive mutating strain growth are groped and experimental study repeatedly, Nutrition and Metabolism characteristic according to bacterial classification, in conjunction with the crop resources advantage, obtained following fermentation culture prescription, make mutant strain can accumulate Avrmectin purpose product well.
1.1 fermentative medium formula: starch 110g, dextrin 20g, soybean cake powder 12g, groundnut meal 8g, yeast powder 10g, cobalt chloride 0.01g, soya-bean oil 5g, yeast extract paste 5g.
Configuration sequence: (1) is measured tap water 600ml earlier and is poured in No. 1 beaker and boil, and weighs 110g starch and breaks into pasty state starch and pour fully gelatinization in the boiling water into; (2) accurately take by weighing cobalt chloride 0.01g with ten thousand/balance and add No. 2 beakers, add water 300ml, put stirring and dissolving on the magnetic stirring apparatus, treat to weigh again after cobalt chloride is fully dissolved soybean cake powder 12g, groundnut meal 8g, yeast powder 10g, dextrin 20g add No. 2 beakers, treat to move into behind the abundant mixing No. 1 beaker; (3) add a spot of water glass stick stirring and dissolving with No. 3 accurate weighing yeast extract paste of small beaker 5g, dissolving back fully adds No. 1 beaker, adds water and is settled to 1000ml, and constantly stir with glass stick, and used material all mixes; (4) transfer PH to 7.5 with acidometer, use the electric furnace heated and boiled, divide while stirring with magnetic stirring apparatus to install in the seed bottle, each seed bottle drips soya-bean oil 3-4 and drips, wrapping back autoclaving (121 ℃ of 22min).
The conventional arrangement method of dividing bottle weighing preparation and transferring PH respectively, advantage is that each fermentation flask nutritive substance is even, but wastes time and energy.But the nutritive substance between every bottle, PH error are bigger.Method of the present invention has overcome the uneven shortcoming of nutritive substance precipitation packing with magnetic stirring apparatus packing while stirring, and each bottle nutritive substance is more accurate, unified simultaneously, and the pH value homogeneous is time saving and energy saving.
1.2 seed flask culture based formulas: dextrin 10g, starch 15g, soybean cake powder 12g, groundnut meal 8g, yeast powder 6g, yeast extract paste 5g, cobalt chloride 0.01g, soya-bean oil 5g.
Configuration sequence: (1) is measured tap water 600ml earlier and is poured in No. 1 beaker and boil, and weighs 15g starch and breaks into pasty state starch and pour fully gelatinization in the boiling water into; (2) accurately take by weighing cobalt chloride 0.01g with ten thousand/balance and add No. 2 beakers, add water 300ml, put stirring and dissolving on the magnetic stirring apparatus, treat to weigh again after cobalt chloride is fully dissolved soybean cake powder 12g, groundnut meal 8g, yeast powder 6g, dextrin 10g add No. 2 beakers, treat to move into behind the abundant mixing No. 1 beaker; (3) add a spot of water glass stick stirring and dissolving with No. 3 accurate weighing yeast extract paste of small beaker 5g, dissolving back fully adds No. 1 beaker, adds water and is settled to 1000ml, and constantly stir with glass stick, and used material all mixes; (4) transfer PH to 7.2 with acidometer, use the electric furnace heated and boiled, divide while stirring with magnetic stirring apparatus to install in the seed bottle, each seed bottle drips soya-bean oil 3-4 and drips, and seals back autoclaving (121 ℃ of 22min).
Compare with original compound method, the unified preparation of nutritive substance, behind the unified accent pH value, packing seed bottle avoids dividing a bottle preparation nutritive substance in the past, transfers pH value, the difference problem of nutritive substance and pH value between each bottle, and time saving and energy saving.
2. the trace element in the nutrition
In order to make pH value constant, must in substratum, add the trace element that can utilize for microorganism, and can make the substratum pH value buffer system of maintenance relatively at the needs that meeting microorganism growth or microorganism accumulation secondary metabolite.Evidence ammonium sulfate is well micro-supplies in nutrition, and ammonium sulfate is decomposed into ammonia and sulfuric acid in culturing process, and ammonia can utilize, and sulfuric acid can fall pH value again.The ammonium sulfate addition is 0.02g in the prescription of above-mentioned substratum.
In A Foman streptomycete gain mutant strain fermentation culturing process, to test and the checking repeatedly of above-mentioned nutrition, the best of finding mutant strain is produced physio-biochemical characteristics such as anti-PH and is compared with the former bacterial classification that sets out and produced certain variation, can impel the induced product accumulation, mutant strain throughput is well expressed.
3. defoamer
In A Foman streptomycete fermentation process, all might produce foam to the decomposition of some composition in the metabolic process of substratum aeration-agitation, A Wei and the substratum etc.Though producing the foam of some amount during the fermentation is normal phenomenon, too much persistence foam is disadvantageous to fermentation.Because foam can occupy the volume of fermentor tank, what influence was ventilated and stirred normally carries out, even causes metabolic disturbance, must eliminate foam.
Therefore, when preparation seed, fermention medium, to add elimination foamy auxiliary material.
4. precursor inductor
After some compound is added into substratum, can directly be attached in biosynthetic process in the product molecule and go, and too big variation does not take place the structure of self, but can improve the output of product, this micromolecular material is called as precursor substance.
In the medium optimization process, through verification experimental verification, in the mutant strain fermentative medium formula, add precursor mass-energy induced product accumulations such as a certain amount of Sodium Propionate, make mutant strain throughput be able to good expression.
Beneficial effect of the present invention is as follows:
1. A Foman streptomyces strain warp
12C
+ 6Select the production bacterial classification of high and stable yields after the heavy ion irradiation mutagenesis again through breeding process provided by the invention, made abamectin fermented tiring stably reach the above height of 5800 μ g/ml, created at present the highest level of industry both at home and abroad.
2. that technology of the present invention has is controlled, adjustable, can repeated characteristics.
3. in conjunction with local resources characteristic, the culture medium cost that provides is low, and aboundresources has remarkable social benefit and economic benefit.
Embodiment
One, preparation substratum
1. fermentation flask substratum
(1) measures tap water 600ml earlier and pour in No. 1 beaker and boil, weigh 15g starch and break into pasty state starch and pour fully gelatinization in the boiling water into;
(2) accurately take by weighing chlorine cobalt 0.01g with ten thousand/balance and add No. 2 beakers, add water 300ml, put stirring and dissolving on the magnetic stirring apparatus, treat to weigh again after cobalt chloride is fully dissolved soybean cake powder 12g, groundnut meal 8g, yeast powder 6g, dextrin 10g add No. 2 beakers, treat to move into behind the abundant mixing No. 1 beaker;
(3) add a spot of water glass stick stirring and dissolving with No. 3 accurate weighing yeast extract paste of small beaker 5g, dissolving back fully adds No. 1 beaker, adds water and is settled to 1000ml, and constantly stir with glass stick, and used material all mixes;
(4) transfer PH to 7.5 with acidometer, use the electric furnace heated and boiled, divide while stirring with magnetic stirring apparatus to install to 250ml beaker (40ml/ bottle), each seed bottle drips soya-bean oil 3-4 and drips, and seals back autoclaving (121 ℃ of 22min).
2. seed flask culture base
(1) measures tap water 600ml earlier and pour in No. 1 beaker and boil, weigh 110g starch and break into pasty state starch and pour fully gelatinization in the boiling water into;
(2) accurately take by weighing chlorine cobalt 0.01g with ten thousand/balance and add No. 2 beakers, add water 300ml, put stirring and dissolving on the magnetic stirring apparatus, treat to weigh again after cobalt chloride is fully dissolved soybean cake powder 12g, groundnut meal 8g, yeast powder 10g, dextrin 20g add No. 2 beakers, treat to move into behind the abundant mixing No. 1 beaker;
(3) add a spot of water glass stick stirring and dissolving with No. 3 accurate weighing yeast extract paste of small beaker 5g, dissolving back fully adds No. 1 beaker, and repeatedly water flushing small beaker is poured washing fluid into large beaker, adds water and is settled to 1000ml, and constantly stir with glass stick, used material all mixes;
(4) transfer PH to 7.2 with acidometer, use the electric furnace heated and boiled, divide while stirring with magnetic stirring apparatus to install to 250ml beaker (40ml/ bottle), each seed bottle drips soya-bean oil 3-4 and drips, and seals back autoclaving (121 ℃ of 22min).
Two, fermentation seed selection
1. warp
12C
+ 6The A Foman streptomyces strain of heavy ion irradiation mutagenesis, irradiation dose is 50Gy, the seed selection of fermenting, leavening temperature (28-0.6) ℃, fermented liquid PH6.5, bottle swingging machine rotating speed 220r/min, inoculation kind of 22 hours ages, inoculum size 4%.
2. warp
12C
+ 6The A Foman streptomyces strain of heavy ion irradiation mutagenesis, irradiation dose is 50Gy, the seed selection of fermenting, 28 ℃ of leavening temperatures, fermented liquid PH7.0, bottle swingging machine rotating speed 250r/min, 24 hours ages, inoculum size 4.5% are planted in inoculation.
3. warp
12C
+ 6The A Foman streptomyces strain of heavy ion irradiation mutagenesis, irradiation dose is 70Gy, the seed selection of fermenting, leavening temperature (28+0.6) ℃, fermented liquid PH7.5, bottle swingging machine rotating speed 260r/min, inoculation kind of 26 hours ages, inoculum size 5%.
In above-mentioned three kinds of embodiment, substratum adds ammonium sulfate 0.02g, and looks foam volume adding defoamer bubble enemy and precursor inductor Sodium Propionate.
Above embodiment all can reach purpose of the present invention.
Claims (5)
1. the height of A Foman streptomyces strain after the heavy ion irradiation mutagenesis breeding process of bacterial classification of tiring, it is characterized by bacterial classification leavening temperature in fermention medium is 28 ± 0.6 ℃, seed culture medium PH 7.0-7.2 fermention medium PH 6.5-7.5, regulate shaking table machine rotating speed 220-260r/min, the inoculation of oxygen solubility and plant 22-26 hour age, the inoculum size 4-5% in the fermented liquid.
2. the tire breeding process of bacterial classification of the height of A Foman streptomyces strain according to claim 1 after heavy ion irradiation mutagenesis, it is characterized by the bottle swingging machine rotating speed is 250r/min.
3. the tire breeding process of bacterial classification of the height of A Foman streptomyces strain according to claim 1 after heavy ion irradiation mutagenesis is characterized by fermention medium and contains following material: starch, dextrin, soybean cake powder, groundnut meal, yeast powder, yeast extract paste, cobalt chloride, soya-bean oil, water.
4. the tire breeding process of bacterial classification of the height of A Foman streptomyces strain according to claim 1 after heavy ion irradiation mutagenesis, the prescription that it is characterized by the fermentation flask fermention medium is: starch 110g, dextrin 20g, soybean cake powder 12g, groundnut meal 8g, yeast powder 10g, cobalt chloride 0.01g, soya-bean oil 5g, water 1000ml.
5. the tire breeding process of bacterial classification of the height of A Foman streptomyces strain according to claim 1 after heavy ion irradiation mutagenesis, the prescription that it is characterized by seed bottle fermention medium is: starch 15g, dextrin 10g, soybean cake powder 12g, groundnut meal 8g, yeast powder 6g, yeast extract paste 5g, cobalt chloride 0.01g, soya-bean oil 5g, water 1000ml.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634471A (en) * | 2012-04-18 | 2012-08-15 | 南京工业大学 | Avermectin B1a high-yielding strain and application thereof |
| CN110387390A (en) * | 2019-06-27 | 2019-10-29 | 华东理工大学青岛创新研究院 | Fermentation medium and zymotechnique |
-
2008
- 2008-12-31 CN CN 200810187967 patent/CN101768586A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102634471A (en) * | 2012-04-18 | 2012-08-15 | 南京工业大学 | Avermectin B1a high-yielding strain and application thereof |
| CN102634471B (en) * | 2012-04-18 | 2013-09-04 | 南京工业大学 | Avermectin B1a high-yielding strain and application thereof |
| CN110387390A (en) * | 2019-06-27 | 2019-10-29 | 华东理工大学青岛创新研究院 | Fermentation medium and zymotechnique |
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Application publication date: 20100707 |