CN100368553C - Production bacterial strain of abamectin with high yield and high secreation rate and new method of extracting AVM - Google Patents
Production bacterial strain of abamectin with high yield and high secreation rate and new method of extracting AVM Download PDFInfo
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- CN100368553C CN100368553C CNB2004100096392A CN200410009639A CN100368553C CN 100368553 C CN100368553 C CN 100368553C CN B2004100096392 A CNB2004100096392 A CN B2004100096392A CN 200410009639 A CN200410009639 A CN 200410009639A CN 100368553 C CN100368553 C CN 100368553C
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Abstract
The present invention relates to a new method for producing abamectin by a high-yield high secretion rate type abamectin producing bacterial strain. The high-yield high secretion rate type abamectin producing bacterial strain used by the new method further mutagenizes deinsectization streptomyces avermitilis (CCTCC NO. M200028) to breed high-titer high secretion rate producing bacterial strain (CCTCC NO. M204069) and bacterial strain (CCTCC NO. M204070). The fermentation titer of the new bacterial strain can be up to 5200 mu g/ml (AVMB1a), which is three times as high as that of original bacterial strain. 60% to 95% of synthesized abamectin is secreted outside bacterial cells, and large numbers of abamectin waxy ball-shaped suspension are formed. In the new method of the present invention, the abamectin in a fermented stock solution is extracted by five steps. Thus, production cost is greatly reduced. In addition, when the abamectin generated by fermentation is used for the production of pesticidal chemical, a technique without extraction can be used.
Description
Technical field
The present invention relates to a kind of novel production bacterial classification of fermentative production Avrmectin, and the supporting with it new abamectin fermented production and the method for extraction.
Background technology
(avermectin AVM) is the macrolide antibiotics that a class is obtained by streptomycete fermentation to Avrmectin.It comprises 8 chemical compositions that structure is very approaching, is called AVMA
1a, AVMA
1b, AVMA
2a, AVMA
2b, AVMB
2a, AVMB
2bIn these 8 components, AVMB
1aPesticide and miticide actility the strongest.Therefore, the Avrmectin product all is AVMB at present both at home and abroad
1aProduct.AVMB
1aThe general chemical pesticide of specific activity of killing to most evil mites and insect on the agroforestry exceeds several times to tens times, and the field using dosage only restrains 1.5 grams for every mu 0.015.This microbiotic is to the useful insect safety of most of kinds, in plant the residence time very short, and very fast in soil be non-toxic substance by microbiological degradation.Avrmectin is acknowledged as a kind of sterilant of microbial source safely and effectively at present.
Avrmectin produces bacterium just synthetic in a large number Avrmectin after mycetocyte grows into certain phase, the AVM that is synthesized almost all is accumulated in the hyphal cell, be not discharged into extracellular (we are called the nonsecreting type bacterial classification), reclaiming Avrmectin must collect sophisticated mycelium with methods such as filter presss earlier, uses a large amount of organic solvent (as acetone, ethanol etc.) extracting then.Yet, with an organic solvent not only increased production cost in large quantities, and direct labor's healthy, work situation and environment protection all brought adverse influence.
Disclose a kind of among the patent documentation CN1294197A by using the novel process of the fermentative production Avrmectin that a small amount of organic solvent reduces cost, this technology is to be based upon on the basis that secretor type AVM produces bacteria strain CCTCC NO:M200028, but M200028 strain fermentation unit is lower, only less than 1500mgAVMB
1a/ L fermented liquid.
Summary of the invention
The present application people carries out the high secretor type bacterial strain of high yield that further mutagenesis screening has obtained some Avrmectins to the M200028 bacterial strain, and mutant strain is used for industrial production through further investigation, has obtained corresponding fermentation and new method for extracting.
The object of the present invention is to provide by the M200028 bacterial strain through ultraviolet ray and nitrosoguanidine mutagenesis and screen the high secretion rate mutant strain of Avrmectin high yield obtain.
Further aim of the present invention provides a kind of the fermentation by the high secretion rate mutant strain of Avrmectin high yield and produces the novel method of Avrmectin.
Another object of the present invention provides a kind of novel method of utilizing simple five steps extraction methods extraction Avrmectin.
The high secretion rate mutant strain of Avrmectin high yield of the present invention belongs to deinsectization streptomycete (Streptomyces avermitilis), be to set out with deinsectization streptomycete LAF-108 (CCTCC NO:M200028) bacterial strain, through ultraviolet and nitrosoguanidine mutagenesis breeding acquisition repeatedly, detailed process is as follows:
1, LAF-108 bacterial strain slant strains is prepared
(prescription is: Fructus Hordei Germinatus powder 5 grams, starch 18 grams, yeast immerse thing 2 grams, saltpetre 1 gram, dipotassium hydrogen phosphate 0.5 gram, sal epsom 0.5 gram, cobalt chloride 0.005 gram, water 1000ml, agar 20-25g, pH7.0-7.2, packing test tube and triangular flask to preparation malt meal starch inorganic salt nutrient agar.121 ℃ of steam sterilizings 30 minutes, pendulum inclined-plane when treating that temperature is reduced to 60 ℃ of left and right sides, the pre-cultivation 2-3 days in the 28-30 ℃ of incubator, streak inoculation LAF-108 then, 27-28 ℃ constant temperature culture 5-7 days.
2, mutagenesis and screening
2.1 luring, ultraviolet gives screening
2.1.1 monospore suspension preparation
The LAF-108 bacterial strain slant strains of ash will be become, with the went out physiological saline of bacterium of 10-12ml spore is washed to come carefully, transfer in the aseptic triangular flask of 250ml that 20-50 grain granulated glass sphere is housed, filled in behind the tampon on shaking table jolting 30 minutes, spore is fully broken up.Add about 20ml stroke-physiological saline solution, mixing again.Filter with the monospore strainer then and obtain monospore suspension.Centrifugal collection monospore, resuspending are in above-mentioned stroke-physiological saline solution, and adjusting spore concentration is 1 * 107/ml.
2.1.2 ultraviolet mutagenesis is handled
Above-mentioned monospore suspension is divided in the little culture dish, uses ultra violet lamp, shone respectively 60 seconds, 90 seconds and 120 seconds, all operate in the darkroom and carry out under the ruddiness.Start magnetic stirrer during irradiation.The postradiation spore suspension of UV suitably dilutes back coating separating plate.
2.1.3 screening
Select on separating plate that the front lawn is abundant, the dark brown steamed bun shape bacterium colony in the back side.The slant medium of transferring again after each bacterium colony number of finishing, intensive line simultaneously is coated with a special agar plate, beats the bacterium piece with punch tool after 10 days, is dipped in the methyl alcohol bubble 24 hours, measures in each bacterial strain vat liquor Avrmectin with the HPLC method and tires.By this method primary dcreening operation, eliminate the low bacterial strain of the plain amount of product more than 70%.Multiple sieve and eventually sieve adopt shake flask culture, survey the total concn of Avrmectin AVMB1a in the concentration of AVMB1a in each strain fermentation supernatant liquor and the fermenation raw liquid with HPLC.Through primary dcreening operation, multiple sieve and whole sieve, selected at last HF-12 bacterial strain carries out nitrosoguanidine (NTG) selection by mutation.
2.2 nitrosoguanidine (NTG) mutagenesis and screening
2.2.1 monospore suspension preparation
With the pH through the 0.1mol/LTris of filtration sterilization and 0.1mol/L maleic acid balanced mix is that 8.0 damping fluid washes HF-12 bacterial strain spore, and this spore suspension is poured in the aseptic triangular flask (interior glaze pearl 30-40 grain).About 30 minutes, filter the acquisition monospore suspension in jolting on the shaking table by aseptic monospore strainer.Centrifugal collection monospore is resuspended in the above-mentioned pH8.0 damping fluid, adjusts spore concentration to 108/ml.
2.2.2 nitrosoguanidine (NTG) mutagenic treatment
Above-mentioned monospore suspension is added in the nitroso guanidine solution, and the ultimate density that makes nitrosoguanidine is 1.0mg/ml.Mutagenic treatment is 60 minutes under the room temperature.The centrifugal nitrosoguanidine of removing at once, again with sterilized water repeatedly behind the centrifuge washing spore 3 times centrifugal collection standby.
2.2.3 separate and screening
Preparation malt meal starch inorganic salt agar plate.Dilution-plate method separates single bacterium colony routinely.Screening method is described with " 2.1.3 ".Screening obtains abamectin fermented height and the also higher deinsectization streptomycete bacterial strain of secretion rate of tiring of 5 strains: AVHF-66, AVHF-31, AVHF-49, AVHF-27 and AVHF-61, make glycerine with mutant strain and guarantee the Tibetan.The fermentation titer of said mutation bacterial strain can reach 5200 μ g/ml (AVMBla), than in patent documentation CN1294197A the fermentation titer of disclosed LAF-108 bacterial strain exceed about 3 times.
These bacterial strains can be secreted into the extracellular with its synthetic Avrmectin, and secretion rate is up to 60%-95%.The Avrmectin that secretion is come out is suspended in the fermented liquid with a kind of form of wax bead, and the content of Avrmectin (AVMB1a) is about 20% in the globe of doing, and this has greatly improved the productive rate of Avrmectin and has reduced extraction cost.
Wherein deinsectization streptomycete AVHF-66 and AVHF-3 1 bacterial strain on September 9th, 2004 in typical culture collection center (Wuhan) preservation of specified depositary institution of State Intellectual Property Office China, preserving number is respectively CCTCC NO:M204069, CCTCC NO:M204070.
Can adopt the conventional substratum of producing Avrmectin and conventional fermentation condition that bacterial strain of the present invention is used for batch fermentation, semicontinuous fermentation or continuously ferment, the Ensure Liquid thing and the continuous mode of production of discharging wax globe promptly can realize continuing to flow.And of the present invention these in batches, semicontinuous or only continuously ferment and to need on the basis of existing batch fermentation processing unit, carrying out local flow improvement and just can realize to equipment.
In the batch fermentation that uses bacterial strain of the present invention to carry out, just above-mentioned globe can be isolated with simple mechanical means after the fermentation ends, use a small amount of organic solvent dissolution again, concentrate with crystallization and can obtain highly purified Avrmectin product.Wherein the usage quantity of organic solvent is less than 15% of existing Avrmectin production technique organic solvent usage quantity.
Use bacterial strain of the present invention no matter to carry out batch fermentation or semicontinuous or continuously ferment and to reduce the consumption of organic solvent significantly; so not only the refinement cost be can reduce, but also direct labor's healthy, labor safety and environment protection helped.Utilize in addition bacterial classification of the present invention carry out AVM continuously or semicontinuous fermentation also improved the production efficiency of plant factor and fermentation greatly.
Can adopt following process carry out bacterial strain of the present invention in batches, semicontinuous or continuously ferment.
Batch fermentation
According to foregoing malt meal starch inorganic salt agar slant culture-medium formulated eggplant bottle inclined-plane.After pre-the cultivation, then do not inoculate any strain in the above-mentioned 5 strain deinsectization streptomycetes if find varied bacteria growing.In 27-29 ℃ of thermostat container, cultivated 5-7 days, treat to receive and keep in refrigerator for fermentation usefulness after the spore maturation.
The seeding tank batch charging coefficient is 60-70%.The seed culture medium composition is: starch 2-4%, soybean cake powder 0.5-1.5%, groundnut meal 0.6-1.2%, active dry yeast powder 0.3-0.7%, corn steep liquor 0.3-0.5%, cobalt chloride 0.0003-0.0015%, defoamer 0.02-0.04% regulates pH to 7.1-7.3 with sodium hydroxide solution before the sterilization.121 ℃ of steam sterilizings, 30 minutes.Treat that substratum is cooled to inoculation above-mentioned slant pore suspension in back about 28 ℃.Inoculum size is 1-2 eggplant bottle of an every 100L seed culture fluid slant strains.At air flow 1: 0.5-1.0,26-28 ℃, 200-400 rev/min stir culture 12-36 hour, thicker to outward appearance, the microscopy mycelia is sturdy, and [this is 2 grades of zymotechniques to culture transferring, if the fermentor tank volume is more than 50 cubic metres to fermentor tank under the multi-branched situation, then can increase first order seed again and cultivate (being three grade fermemtation technology), the composition and the culture condition of substratum are the same].
The composition of fermentation cylinder for fermentation substratum is: starch 6-10%, active dry yeast powder 1.0-1.4%, soybean cake powder 0.5-2.0%, Semen Maydis powder 0.5-1.2%, Repone K 0.2-0.6%, potassium primary phosphate 0.03-0.05%, cobalt chloride 4-6 μ g/ml (final concentration), sal epsom 0.03-0.05%, lime carbonate 0.1-0.3%, fire resistant alpha-diastase 0.004-0.01%, and add the mixing defoamer 0.01-0.05% that contains bubble enemy and vegetables oil in advance, be settled to volume requiredly with tap water, preceding pH6.8-7.0 disappears.Behind steam sterilizing, inoculate, inoculum size is 2-10%, in warm 27-29 ℃ in material, air flow 1: 0.8-1.15, cultivated 9-10 days under the stirring velocity 150-350 rev/min of condition, pH rises to more than 7.5, the obvious self-dissolving of mycelia, when the B1a component of Avrmectin reaches peak-peak, open and put tank valve, emit whole fermented liquids, sieve the AVM bead of getting in the fermentor tank with wet screening machinery (screen cloth is the 40-80 order), globe enters extractor behind centrifuge dripping, with 2-4 times of volumes toluene (or ethyl acetate) dissolving, decolorizing with activated carbon, anhydrous sodium sulfate dehydration, destainer concentrates, and adds the AVMB1a crystal seed at last and obtains the AVMB1a crystallization.
Semicontinuous fermentation
As described in patent documentation CN1294197A, a discharge orifice is opened in the place of high about 65-75% in fermentor tank right cylinder side, and corresponding pipeline valve is installed, so that discharge the AVM globe in the fermented liquid, and can lead to steam and sterilizes.
The process that slant strains is prepared and seed liquor prepares is described with above batch fermentation.The fermention medium of semicontinuous fermentation is formed: starch 6-10%, active dry yeast powder 1.0-1.4%, Semen Maydis powder 0.5-1.5%, soybean cake powder 0.5-2.0%, corn steep liquor 0.2-1.0%, Repone K 0.2-0.6%, potassium primary phosphate 0.03-0.06%, cobalt chloride 4-6 μ g/ml (final concentration), sal epsom 0.03-0.05%, lime carbonate 0.1-0.3%, fire resistant alpha-diastase 0.004-0.01%, and add the mixing defoamer 0.01-0.05% of bubble enemy and vegetables oil, and be settled to volume requiredly with tap water, preceding pH6.8-7.2 disappears.After 130-150 ℃ of steam is even eliminated bacterium, lower the temperature and enter fermentor tank and inoculate.Inoculum size is 2-10%, in warm 27-28 ℃ in material, and air flow 1: 1-1.15, stirring velocity 150-350 rev/min of condition bottom fermentation cultivated.After fermentation proceeds to 120 hours, the total reducing sugar, reducing sugar, amino nitrogen and the Avrmectin B1a component concentration that detected in the one time fermentation liquid every 4-8 hour.When being lower than 1%, reducing sugar begins to mend sugar.The nutrient solution prescription that is replenished is: glucose 5-30%, vegetables oil (being preferably soya-bean oil and peanut oil) 1-10%.Since microscopy mycelia aging in every 6-8 hour in the 9th day whether.If there is aging phenomenon then in stream Ensure Liquid liquid, to increase an amount of yeast extractive substance.Each sampling detection fermented liquid upper strata contained globe part A VMla content since the 9th day, and the abamectin Bla component concentration is higher than 2000 μ g/ml, and then the short period of time stops to stir and ventilation, opens above-mentioned discharge orifice valve simultaneously, discharges the plain liquid that contains on fermented liquid upper strata.The amount of fermentating liquid volume of at every turn emitting and the fresh medium that replenishes is equivalent to the 5-10% of original fermented solution volume.
By feed supplement, replenish fresh culture and suitably adjust fermentation condition, prolong above fermenting process as far as possible, improve the Avrmectin ultimate production.The fermented liquid of discharging from last discharge orifice is gathered in the crops the AVMla bead through the wet screening machine at every turn, enter extractor after the drying, toluene or ethyl acetate with 2-4 times of volume are dissolved, and solution filtering and dehydration back concentrate 1-2 and extraordinarily go into the AVMBla crystal seed, obtain the Avrmectin crystallization.
After some days, produce bacterial strain and occur old and feeble self-dissolving inevitably, if the obvious old and feeble self-dissolving of mycelia then can be put jar.Putting a jar back AVMBla extracting method is the following five-step approach that will narrate.
Continuously ferment
Carry out scrap build according to two or more isometric(al)s (or not co-content) placed in-line continuous fermentation process of fermentor tank.A discharge orifice is opened in the place of high about 65-75% in fermentor tank right cylinder side, and corresponding pipeline valve is installed.Establish material-feeding port on each jar after No. 2 jars, corresponding pipeline valve is installed at discharge orifice.
According to foregoing malt meal starch inorganic salt agar slant culture-medium formulated eggplant bottle or test tube slant.The pre-cultivation after 3 days then do not inoculated any strain in the above-mentioned 5 strain deinsectization streptomycetes if find varied bacteria growing.In 27-29 ℃ of thermostat container, cultivated 5-7 days, treat to take out after the spore maturation, be stored in 4 ℃ of refrigerators.Carry out species test with shaking bottle mode before last jar, the conclusive evidence bacterial classification is not used for fermentative production after having living contaminants.
The culture medium prescription of liquid seeds is described with batch fermentation, and fermention medium is identical with the semicontinuous fermentation substratum.Fermention medium is by continuous sterilization system sterilization, is stored in the fermention medium basin after cooling the temperature to 28-30 ℃.
The dilution range that adopts in the continuous fermentation process is 0.005-0.03 (l/h), and the series can number is 3 or 4, volumetric ratio: 1: 1: 1 (or 1: 1: 1: 1).Temperature: No. 1 jar and No. 2 jars are 27-28 ℃, and No. 3 jars (or No. 3 jars and No. 4 jars) are 26-27 ℃; The pH::1 jar is 6.4-6.8, and No. 2 jars (or No. 2 jars and No. 3 jars) are 5.6-6.5, and No. 3 jars (or No. 3 jars and No. 4 jars) are 6.0-6.8; Air quantity: No. 1 jar is 1: 1.0-1.5, No. 2 jars (or No. 2 jars and No. 3 jars) are 1: 1.0-1.2, No. 3 jars (or No. 3 jars and No. 4 jars) are 1: 0.8-1.0.
No. 1 jar stirs and often opens, and 2 and No. 3 jars (or 2 and 3 and No. 4 jars) churning time looks the dissolved oxygen situation is often opened or intermittently open stirring.Other fermentation condition is with reference to semicontinuous fermentation, and being in due course, stream adds nutriment such as glucose, vegetables oil, yeast extractive substance in jar.Jar in the end, i.e. 3 or No. 4 jar discharges, marker method can carry out with reference to semicontinuous fermentation.Also can discharge sophisticated fermented liquid evenly incessantly.
The fermented liquid that contains the AVM bead of discharging is sieved coccoid thing also directly enter extractor after the drying.Extract solvent with toluene or ethyl acetate, decolorizing with activated carbon, concentrating under reduced pressure behind the anhydrous sodium sulfate dehydration (temperature is between 50-65 ℃) adds the AVMBla crystal seed and carries out crystallization, can obtain the Avrmectin crystal.
After continuously fermenting some days, produce the same semicontinuous fermentation of treatment process after catabiosis generally appears in bacterial strain.
AVMBla five-step approach extraction process
The present invention uses bacterial classification (AVHF-66, AVHF-31) to obtain containing in a large number the fermenation raw liquid of the wax shape bead of AVMBla by above-mentioned fermentation process, the contriver is reduced to following " five-step approach " with the former extraction process process of AVMBla then, thereby has saved a large amount of organic solvents and reduced the investment of refining equipment." five-step approach " extraction process is described in detail as follows:
1. collect AVM wax bead by screening from fermenation raw liquid
A large amount of oyster whites that suspending in the fermenation raw liquid that utilizes strain fermentation such as AVHF-31 to obtain are to xanchromatic wax shape bead, and these beads are that oozy Avrmectin aggegation forms from production bacterium cells such as AVHF-31.This bead hydrophobicity is stronger, and a material diameter is easy to the way of screening itself and mycelium and water be separated more than 0.5mm.Screening selects for use vibratory screening apparatus or rotating screen all can.Screen cloth 40-80 order stainless (steel) wire.During screening the pipeline of running water pipe and conveying fermented liquid is all guided on the screen cloth, Yi Bian promptly put fermented liquid, Yi Bian wash screen cloth lentamente with tap water.AVM wax bead can be collected in the screen cloth top, contains a large amount of mycelium in the water of screen cloth below.The mycelium suspension enters disc centrifuge or sheet frame, and the mycelium of recovery can be produced the agricultural chemicals that kills soil nematodes or make antibiotic fertilizer.
2. centrifuge dripping AVM wax bead
Dry wet AVM bead with link-suspended basket centrifuge, be lined with the stainless (steel) wire of 100 orders (or 80 orders) in the link-suspended basket centrifuge, this net can disassemble easily, so that collect the AVM bead and clean mesh.
3. acetic acid ethyl dissolution AVM wax bead
The AVM bead acetic acid ethyl dissolution that dries, the color of looking lysate determines whether to add bleaching agent bleaching.Lysate is dewatered with anhydrous sodium sulphate, obtain the liquid that dewaters.
4. thickening liquid
Dehydration liquid concentrates with vacuum concentration pan, 50 ℃-60 ℃ of thickening temperatures, and concentrating vacuum tightness is-0.09Mpa to be concentrated into AVMBla concentration and to reach more than 5%.
5. crystallization
Carry out crystallization in crystallizer, crystal seed is the AVMBla crystal of good crystalline, and consumption is 0.05%-0.1% (w/v).Crystal solution is that sherwood oil adds dehydrated alcohol, and the two volume ratio is 85: 15.
The agricultural chemicals hands-free sweetening process of Avrmectin raw material
The preparation agricultural chemicals is increasing with the amount of required Avrmectin at present, has become the market of the former medicine maximum of AVM.Use strain fermentation of the present invention and produce AVM, the raw material that can produce the Avignon medicine according to a conventional method (is about to crystalline mother solution simmer down to ointment, with the raw materials for production of ointment as the AVM agricultural chemicals), also can be by the special production technique of the present invention---" hands-free sweetening process " direct production.The production process of this method is that fermenation raw liquid is sifted out wherein AVM wax bead with wet screening, and is air-dry with 40 ℃ of left and right sides hot blasts after drying through link-suspended basket centrifuge, through content detection, sealing shading packing for producing the raw material of Avignon medicine.
Description of drawings
Fig. 1 is the synoptic diagram of fermentor tank continuous mode and Flow of Goods and Materials in the continuous fermentation process of the present invention.
Wherein:
1, connects steam manifold
2, connect the air total filter
3, substratum preparing tank
4, vapor filter
5, air pre cleaner
6, air fine filter
7, seeding tank
8, No. 1 fermentor tank
9, No. 2 fermentor tanks
10, No. 3 fermentor tanks
11, continuous sterilisation tower
12, keep jar
13, plate-type heat exchanger
14, temperature is reduced to the no bacteria fermentation culture medium about 30 ℃
15, feed supplement jar
16, the mixed solution that contains Avrmectin wax globe and fermented liquid of emitting from last discharge outlet
17, connect wet screening machine (screen cloth 40-80 order)
Fig. 2 is an AVMBla five-step approach extraction process schematic flow sheet of the present invention.
[embodiment]
To further illustrate the present invention in following examples, these embodiment only are used to illustrate the present invention, but do not constitute any limitation of the invention.
Embodiment 1
Use deinsectization streptomycete AVHF-31 bacterial strain to produce Avrmectin by batchwise.
According to foregoing malt meal starch inorganic salt agar slant culture-medium formulation inclined-plane.Through 3 days pre-cultivations, then do not inoculate the AVHF-31 bacterial strain if find varied bacteria growing.In 28 ℃ of thermostat containers, cultivated 5 days, treat to collect after the spore maturation, be stored in 4 ℃ of refrigerators and use for fermentation.
Prepare the 10L seeding tank, batch charging coefficient 65%.The seed culture medium composition is: starch 3%, and soybean cake powder 0.8%, groundnut meal 0.9%, yeast extract paste 0.45%, yeast powder 0.45%, corn steep liquor 0.3%, cobalt chloride 0.003%, defoamer 0.06% is regulated pH to 7.2 with sodium hydroxide solution before the sterilization.121 ℃ of steam sterilizings 30 minutes treat that substratum is cooled to the spore suspension of inoculation AVHF-31 bacterial strain after 28 ℃.Inoculum size is 2 test tube slant seeds of every jar of seed culture fluid (6.5L) inoculation.Air flow 1: 0.8,27 ℃, 350 rev/mins of agitation conditions were cultivated 36 hours down, and thicker to outward appearance, the microscopy mycelia is sturdy, multi-branched, and culture transferring is to the 100L fermentor tank under the no living contaminants situation.
The fermentation cylinder for fermentation substratum is: starch 10%, active dry yeast powder 1.0%, Semen Maydis powder 1%, soybean cake powder 1.5%, Repone K 0.6%, potassium primary phosphate 0.03%, cobalt chloride 5 μ g/ml (final concentration), sal epsom 0.03%, lime carbonate 0.3%, fire resistant alpha-diastase 0.01%,, and add the mixing defoamer 0.01% that contains bubble enemy and vegetables oil in advance, be settled to 70L with tap water, batch charging coefficient 70%, preceding pH7.0 disappears.Inoculation after sterilizing, inoculum size is 9.3%, 28 ℃ of jar temperature, air flow 1: 0.8, stirring velocity is cultivated down for 230 rev/mins, when reducing sugar content is lower than 1.0% in the fermented liquid, adds benefit sugar by feed supplement jar stream.Cultivate altogether about 12 days.When pH rises to more than 7.5, the obvious self-dissolving of mycelia when the Bla component of Avrmectin reaches peak-peak, is opened and is put tank valve, emits whole fermented liquids, with the wax globe (1400 gram) of wet screening results AVM.With 4000ml acetic acid ethyl dissolution globe, decolorizing with activated carbon, anhydrous sodium sulfate dehydration, vacuum concentration adds the AVMBla crystal seed at last and obtains Avrmectin crystallization 282.3 grams, wherein Bla component 253 grams then.
Embodiment 2
Use deinsectization streptomycete AVHF-66 bacterial strain to produce Avrmectin by batchwise.
According to foregoing malt meal starch inorganic salt agar slant culture-medium formulation inclined-plane.Through 3 days pre-cultivations, then do not inoculate the AVHF-66 bacterial strain if find varied bacteria growing.In 27 ℃ of thermostat containers, cultivated 7 days, treat to collect after the spore maturation, be stored in 4 ℃ of refrigerators and use for fermentation.
Prepare the 10L seeding tank, batch charging coefficient 65%.The seed culture medium composition is: starch 4%, and soybean cake powder 0.5%, groundnut meal 1.2%, active dry yeast powder 0.3%, corn steep liquor 0.5%, cobalt chloride 0.0015%, defoamer 0.04% is regulated pH to 7.2 with sodium hydroxide solution before the sterilization.121 ℃ of steam sterilizings 30 minutes treat that substratum is cooled to the spore suspension of inoculation AVHF-66 bacterial strain after 28 ℃.Inoculum size is 3 test tube slant spores of every jar of seed culture medium (6.5L) inoculation.Air flow 1: 1.0,27 ℃, 350 rev/mins of agitation conditions were cultivated 24 hours down, and thicker to outward appearance, the microscopy mycelia is sturdy, multi-branched, and culture transferring is to the 100L fermentor tank under the no living contaminants situation.
The fermentation cylinder for fermentation substratum is: starch 11%, active dry yeast powder 1.4%, Semen Maydis powder 1%, soybean cake powder 1.0%, peanut soybean cake powder 0.5%, Repone K 0.2%, potassium primary phosphate 0.05%, cobalt chloride 5 μ g/ml (final concentration), sal epsom 0.05%, lime carbonate 0.2%, fire resistant alpha-diastase 0.0004%, and add the mixing defoamer 0.06% that contains bubble enemy and vegetables oil in advance, and being settled to 70L with tap water, preceding pH7.2 disappears.Inoculation after sterilizing, inoculum size is 9.0%, at 28 ℃ of jar temperature, air flow 1: 1.8, stirring velocity was cultivated 12 days down for 250 rev/mins, when pH rises to more than 7.5, the obvious self-dissolving of mycelia is when the B1a of Avrmectin reaches peak-peak, put tank valve at the bottom of opening jar, blowing limit, limit wet screening, mesh size is 80 orders, collects AVM globe 1328.6 grams (dry weight) that are wax.The content of Avrmectin is 21% in dried globe.Use the 4000ml acetic acid ethyl dissolution, decolorizing with activated carbon, anhydrous sodium sulfate dehydration is concentrated into about 1200 milliliters, adds crystal seed at last and obtains Avrmectin crystallization 279.1 grams, wherein Bla component 251.2 grams.Feed supplement method and main technologic parameters are seen embodiment 1.
Employing and embodiment 1 identical step just replace with AVHF-27, AVHF-49 respectively with the AVHF-31 bacterial strain and the AVHF-61 bacterial strain is produced Avrmectin by batchwise.
The output of these three bacterial classifications is as shown in table 1:
Table 1 AVHF-27, AVHF49 and three bacterial strain batch fermentations of AVHF-61 AVMB
1aOutput
| Embodiment | Bacterial strain | Avrmectin (gram) | Avrmectin B 1a(gram) |
| |
AVHF-27 | 266.5 | 242.5 |
| Embodiment 4 | AVHF-49 | 267.4 | 238.8 |
| |
AVHF-61 | 270.8 | 251.8 |
Comparing embodiment
Use deinsectization streptomycete LAF-108 bacterial strain (being the bacterial classification CCTCC NO:M200028 among the patent documentation CN1294197A) to produce Avrmectin by batchwise.
According to foregoing malt meal starch inorganic salt agar slant culture-medium formulation inclined-plane.Through 3 days pre-cultivations, then do not inoculate the LAF-108 bacterial strain if find varied bacteria growing.In 28 ℃ of thermostat containers, cultivated 6 days, treat to collect after the spore maturation, be stored in 4 ℃ of refrigerators and use for fermentation.
The seed preparation process is with embodiment 1.
The fermentation cylinder for fermentation substratum is: starch 9%, active dry yeast powder 1.2%, Semen Maydis powder 1%, Repone K 0.4%, potassium primary phosphate 0.04%, cobalt chloride 5 μ g/ml (final concentration), sal epsom 0.04%, lime carbonate 0.2%, fire resistant alpha-diastase 0.005%, and add the mixing defoamer 0.02% that contains bubble enemy and vegetables oil in advance, be settled to 70L with tap water, batch charging coefficient 70%, preceding pH7.0 disappears.Inoculation after sterilizing, inoculum size is 8.5%, 28 ℃ of jar temperature, air flow 1: 0.9, stirring velocity is cultivated down for 250 rev/mins, and after fermentation 3 days, reducing sugar content is low 1% o'clock in the fermented liquid, adds glucose by feed supplement jar stream and makes it to more than 1.3%.When pH rises to more than 7.5, the obvious self-dissolving of mycelia when the Bla component of Avrmectin reaches peak-peak, is opened and is put tank valve, emits fermented liquid, by the wax globe of wet screening machine results AVM.The content of Avrmectin is 20% in dried globe.With 3300ml vinyl acetic monomer dissolving globe, decolorizing with activated carbon, destainer are again through anhydrous sodium sulfate dehydration, and concentrating under reduced pressure removes half volume, add the AVMBla crystal seed while hot and slowly lower the temperature to obtain Avrmectin crystallization 65.0 grams, wherein Bla component 58.5 grams.
Use deinsectization streptomycete AVHF-31 bacterial strain to press semi-continuous type and produce Avrmectin.
Open a discharge orifice in high about 70% the place of jar, 100L fermentor tank right cylinder side, and corresponding pipeline valve is installed.
Prepare and seed liquor as preparation slant strains as described in the embodiment 1.The fermention medium of preparation semicontinuous fermentation: starch 8%, 12 liters in active dry yeast powder 1.2% or fresh yeast mud, Semen Maydis powder 1.0%, soybean cake powder 1.2%, Repone K 0.4%, potassium primary phosphate 0.05%, cobalt chloride 5 μ g/ml (final concentration), sal epsom 0.04%, lime carbonate 0.3%, fire resistant alpha-diastase 0.005% adds the mixing defoamer that contains bubble enemy and vegetables oil by 0.06% of culture volume, be settled to 70L with tap water, preceding pH7.2 disappears.After even eliminating bacterium, 140 ℃ of steam enter fermentor tank, be cooled to 28 ℃ of inoculations, inoculum size is 10%, 28 ℃ of jar temperature, air flow 1: 1.1,250 rev/mins of bottom fermentations of stirring velocity are cultivated, after fermentation proceeds to 48 hours, and the total reducing sugar, reducing sugar, amino nitrogen and the abamectin Bla component concentration that detected in the one time fermentation liquid every 8 hours.When being lower than 1.0%, reducing sugar begins feed supplement.The nutrient solution prescription that is replenished is: glucose 10%, vegetables oil (soya-bean oil) 1%.Whether the microscopy mycelia aging of taking a sample since the 9th day per 8 hours.If there is aging phenomenon then in stream Ensure Liquid liquid, to increase an amount of yeast extractive substance, potassium primary phosphate and corn steep liquor.Since the 9th day, supernatant liquor part Avrmectin B1a component concentration in the fermented liquid of at every turn taking a sample to check.When the HPLC method recorded Avrmectin B1a component concentration and is higher than 2500 μ g/ml, the short period of time stopped to stir and ventilation, opens above-mentioned discharge orifice valve simultaneously, discharges the AVM wax globe that floats over the fermented liquid upper strata, common about 10 liters of this globe and water.Wet screening obtains wax globe 174 grams, and wherein the content of Avrmectin is 18.5%.Then, add 10 liters of fresh mediums, 24 hours repeat above-mentioned fermenting process once, carry out altogether 12 times, and 12 common acquisitions contain plain wax 3450.8 grams.Obvious old and feeble self-dissolving appears in mycelia after 21 days, puts jar this moment.Each globe that obtains directly enters extractor, dissolves filtration then with the vinyl acetic monomer of 3 times of volumes, the decolouring dehydration concentrates, and adds Avrmectin B1a crystallization at last, obtain the Avrmectin crystallization, obtain Avrmectin 441.1 grams altogether, wherein obtain abamectin Bla component 397.0 grams.
Press the technology of present embodiment, the 100L fermentor tank obtained 461.1 gram Avrmectins in 20 days, obtained 182.3 grams than 11 days of embodiment 1 and had improved working efficiency.The consumption ratio of organic solvent and embodiment 1 are similar.
Four bacterial strains such as use deinsectization Streptomycin A VHF-66 are pressed semi-continuous type and are produced Avrmectin.
Adopt and embodiment 6 identical steps, just the AVHF-31 bacterial strain is replaced with AVHF-66, AVHF-27, AVHF49 and AVHF-61 bacterial strain respectively, the crystallization output of the Avrmectin that obtains at last sees Table 2:
22 days AVMB of 4 bacterial strain semicontinuous fermentations such as table 2 AVHF-66
1aUltimate production
| Embodiment | Bacterial strain | Avrmectin (gram) | Avrmectin B 1a(gram) |
| Embodiment 7 | AVHF-66 | 700.5 | 644.3 |
| Embodiment 8 | AVHF-27 | 651.4 | 579.8 |
| Embodiment 9 | AVHF-49 | 609.7 | 544.6 |
| | AVHF-61 | 655.9 | 612.2 |
Use deinsectization streptomycete AVHF-31 bacterial strain to press continuous fermentation method and produce Avrmectin.
Fermentor tank is transformed with embodiment 6, the right cylinder position No. 2 and No. 3 fermentor tank the same sides, and high about tank body total height 70% place opens a discharge orifice, and establishes the feed supplement product on No. 2 and No. 3 fermentor tanks, at last discharge outlet and the corresponding pipeline valve of feed supplement product installation.Mode of connection between 3 jars as shown in Figure 1
The culture medium prescription of liquid seeds is with embodiment 1, and fermention medium is with semicontinuous fermentation substratum (seeing embodiment 6).Fermention medium is by continuous sterilization system sterilization, is stored in the fermention medium basin after cooling the temperature to about 28 ℃.
The extent of dilution that this continuous fermentation process is taked is 0.01 (l/h), and the series can number is 3, and the working capacity of each jar is 100L, volumetric ratio: 1: 1: 1.Temperature: No. 1 jar and No. 3 jars are 28 ℃, and No. 3 jar is 26 ℃; The pH:1 jar is that 6.8, No. 2 jars are that 6.5, No. 3 jars are 6.8; Air quantity: No. 1 jar is 1: 1.2, and No. 2 jar is 1: 1.0, and No. 3 jar is 1: 0.8.No. 1 jar stirs and often opens, and 2 and No. 3 jar churning time are looked the dissolved oxygen situation is often opened or intermittently opened.Stirring velocity is 250 rev/mins.With reference to the parameter that embodiment 6 provides, stream adds glucose, vegetables oil, yeast extractive substance in jar when the needs feed supplement, also can discharge sophisticated fermented liquid evenly incessantly.The content of Avrmectin is 19% in globe.After so continuously fermenting 22 days, catabiosis generally appears in the cell of producing bacterium, stops fermentation and puts jar.
Collect the coccoid thing of AVM wax with wet screening, produce the AVMBla crystallization with reference to embodiment 1, obtain Avrmectin crystal 1162.3 grams altogether, on average each jar produces Avrmectin 17.6 grams every day.
Press the technology of present embodiment, every jar of output AVMBla crystallization every day 17.6 gram that continuously ferments, and among the embodiment 1 every jar of (batch fermentation) same bacterial classification average every day only produce AVMBla crystallization 16.0 grams, the efficient of continuously fermenting improves 10%.The consumption ratio of organic solvent and embodiment 1 are similar.
Use deinsectization Streptomycin sulphate and AVHF-66, AVHF-27, AVHF-49 and AVHF-61 bacterial strain to press continuous fermentation method and produce Avrmectin.
Adopt and embodiment 11 identical steps, just the AVHF-31 bacterial strain is replaced with AVHF-66, AVHF-27, AVHF-49 and AVHF-61 bacterial strain respectively, the crystallization output of the Avrmectin that obtains at last sees Table 3:
4 bacterial classification AVMB in the process of continuously fermenting such as table 3 AVHF-66
1aEvery jar every day output
| Embodiment | Bacterial strain | Avrmectin crystal yield (AVMB 1aGram/jar/sky) |
| |
AVHF-66 | 18.9 |
| |
AVHF-27 | 17.1 |
| |
AVHF-49 | 17.4 |
| |
AVHF-61 | 18.0 |
The 10M3 ferment tank obtains fermenation raw liquid 8.0M3, fermentation titer (AVMB1a) 4200 μ g/ml, AVMB1a secretion rate 90% after finishing.Theoretical Calculation is produced bacterial classification and is secreted into extracellular AVMB1a and is 30.24 kilograms altogether as can be known.These AVMBla are suspended in the fermented liquid with the small-particle that other component of AVM forms some waxs.Extract these AVMBla and form crystallization and be divided into following 5 steps:
The first step wet sieving from
Fermenation raw liquid is cooled to below 25 ℃ in the fermented liquid basin, and tank pressure rises to 0.15MPa.Opening baiting valve guides to fermenation raw liquid on the wet screening machine by pipeline.The screen cloth of wet screening machine is 60 purpose Stainless Steel Cloths.Meanwhile open the running water pipe valve on the wet screening machine, regulate the flow velocity of fermented liquid and the flow velocity of tap water and arrive suitably.When the AVM particle will be filled in the screen cloth, change screen cloth, collect the AVM wax wet granular that sifts out.From this jar fermented liquid, sieve altogether wet granular 199.6kg.
The second step wax particle dries
Wet granular 199.6kg adds water 600kg, in mixing tank with 50 rev/mins mixing speed resuspending.Utilize air pressure that the wet granular of resuspending is piped to link-suspended basket centrifuge.Be lined with a special nylon yarn string bag in this link-suspended basket centrifuge, the gauze mesh is 120 orders.Centrifugal rotational speed 2000rpm.Blowing limit, limit is centrifugal, stops charging when particle in the string bag is expired soon.Stop to continue centrifugal 5 minutes after the charging.Shut down, take out the AVM wax bead that dries.The coccoid thing 89.2kg of AVM that results dry.
The particle dissolving of the 3rd step
The AVM wax particle that in dissolving vessel, has dried with the dissolving of second vinegar ethyl ester.The acetic acid ethyl ester consumption is 20: 1 (v/w), and promptly 1 kilogram of above-mentioned AVM particle adds 20 liters of ethyl acetate and dissolves, and dissolve the 6% adding anhydrous sodium sulphate powder of back by gross weight substantially, limit edged stirring (120rpm).If solution colour can add powdered activated carbon decolouring (the normal jar of fermentation is without decolorizing with activated carbon) by 8% (w/w) more deeply.Continue to stir and heat, when solution temperature reaches 50 ℃, stop to heat to solution.While hot this solution is filtered with small-sized stainless steel sheet frame, remove undissolved solid matter.This batch obtains 1.7 tons of filtrates, and AVMBla content is 1.74% in the filtrate.
The 4th step filtrate concentrates
In vacuum concentrator, filtrate is concentrated 15 times under 55 ℃, vacuum tightness pact-0.09MPa condition.
The 5th step crystallization
Join crystal solution.It is that 90-120 ℃ sherwood oil and dehydrated alcohol mixes that crystal solution consists of boiling range, and the two ratio is a sherwood oil: ethanol=85: 15 (v/v).Be warming up to 55 ℃ after in crystallizer, mixing.The concentrated solution that the 4th step was carried slowly adds crystallizer, and the limit edged stirs, mixing speed 50rpm.Can add an amount of AVMBla crystal seed, amount of seed 1% (w/w) after adding concentrated solution.With the jar temperature of warm water crystallization control jar, on average per hour following 1 ℃ of temperature is expected in order, keeps 12 hours in the time of 2 ℃-4 ℃.Crystallization is finished after link-suspended basket centrifuge is collected crystal, and the precipitation drying detects AVMBla content.This batch obtains crystallization 21.89kg altogether, and wherein B1a content is 92%.
Claims (5)
1. one kind is utilized deinsectization streptomycete (Streptomyces avermitilis) to ferment to obtain to contain the fermenation raw liquid of AVM wax bead and adopts five step extraction methods to extract and produce AVMB
1aMethod, it is characterized in that described five the step extraction methods comprise step:
A. collect AVM wax bead by screening from fermenation raw liquid;
B. centrifuge dripping AVM wax bead;
C. dissolve AVM wax bead;
D. thickening;
E. add AVMB
1aCrystal seed carries out crystallization.
2. according to the process of claim 1 wherein that described fermentation is batch fermentation or semicontinuous fermentation.
3. according to the process of claim 1 wherein that described fermentation is for continuously fermenting.
4. in step c, use acetic acid ethyl dissolution AVM wax bead according to the process of claim 1 wherein.
5. according to the method for claim 1 or 4, wherein the crystal solution of using in step e is 85: 15 sherwood oil and dehydrated alcohol as 90 ℃～120 ℃ of boiling ranges and volume ratio.
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| CN100372462C (en) * | 2006-09-06 | 2008-03-05 | 云南大学 | Microbiological agent resisting rice bakanae disease, preparation method and application thereof |
| CN102634471B (en) * | 2012-04-18 | 2013-09-04 | 南京工业大学 | Abamectin B1a high-yield strain and application thereof |
| CN103039534B (en) * | 2012-12-28 | 2014-11-05 | 李峰 | Microbial preparation for inhibiting root-knot nematode and preparation method and application 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 |
| CN104876991B (en) * | 2015-06-12 | 2018-01-12 | 齐鲁制药（内蒙古）有限公司 | A kind of method that secondary crystallization prepares Avermectin B1a fine powder in Avermectin B1a crystalline mother solution |
| CN108060193B (en) * | 2018-01-31 | 2021-08-10 | 天俱时工程科技集团有限公司 | Production process of abamectin |
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| CN1281900A (en) * | 2000-07-25 | 2001-01-31 | 高东卫 | Extraction method of abamectin |
| CN1294197A (en) * | 2000-10-30 | 2001-05-09 | 武汉绿世纪生物工程有限责任公司 | Process for preparing evericin by fermentation method |
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| CN1281900A (en) * | 2000-07-25 | 2001-01-31 | 高东卫 | Extraction method of abamectin |
| CN1294197A (en) * | 2000-10-30 | 2001-05-09 | 武汉绿世纪生物工程有限责任公司 | Process for preparing evericin by fermentation method |
Non-Patent Citations (1)
| Title |
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| 阿维菌素的工业化生产研究. 宋渊.精细与专用化学品，第21期. 2002 * |
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