CN1294197A - Process for preparing evericin by fermentation method - Google Patents
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- CN1294197A CN1294197A CN 00130233 CN00130233A CN1294197A CN 1294197 A CN1294197 A CN 1294197A CN 00130233 CN00130233 CN 00130233 CN 00130233 A CN00130233 A CN 00130233A CN 1294197 A CN1294197 A CN 1294197A
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Abstract
A process for preparing evericin by fermentiation method features that a microbe which can generate evericin and can secret the evericin to outside of cells is used in the fermenting procedure. Its advantages are high safety and environmental protection.
Description
The present invention relates to a kind of novel process of fermentative Production Avrmectin.
Avrmectin (avermectin, AVM) be a kind of macrolide antibiotics that obtains by streptomycete fermentation, it has good killing action to more than ten purpose hundreds of kind insect in agricultural and the forestry, insecticidal activity exceeds a few to tens of times than general chemical pesticide, and the field using dosage only is every mu 0.015 to 1.5 gram.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.Because the advantage that these of Avrmectin are outstanding, it is acknowledged as a kind of the most microbe-derived sterilant.
Yet, it is just synthetic in a large number Avrmectin after mycetocyte grows into certain phase that Avrmectin produces bacterium, and this material that is synthesized almost all is accumulated in the hyphal cell, be not discharged into the extracellular, reclaiming this material must collect sophisticated mycelium with methods such as filter presss earlier, uses a large amount of organic solvent (as acetone, ethanol etc.) extracting then.With an organic solvent not only increased production cost in large quantities, and direct labor's healthy, labor safety and environment protection brought adverse influence.
The novel process that the purpose of this invention is to provide the low fermentative Production Avrmectin of the few production cost of a kind of consumption of organic solvent.
The inventor has found some to produce the secretor type bacterial strain of Avrmectin through further investigation, can produce Avrmectin and Avrmectin is secreted into the outer microorganism of hyphal cell thereby use during the fermentation, and finish the present invention whereby.
More than said microorganism belong to A Fumai streptomycete (claiming deinsectization streptomycete again) (Streptomyces avermitilis), be from A Fumai streptomycete A-9 bacterial strain, through ultraviolet ray and nitrosoguanidine mutagenesis breeding acquisition, detailed process is as follows:
1, A-9 bacterial strain slant strains is prepared
(prescription is preparation malt meal starch inorganic salt nutrient agar: Fructus Hordei Germinatus powder 5 grams, starch 18 grams, yeast extract 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, pH 7.0-7.2), packing test tube and triangular flask.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 A-9 bacterial strain spore then, 27-28 ℃ constant temperature culture 5-7 days.
2, mutagenesis and screening
2.1 ultraviolet mutagenesis and screening
2.1.1 monospore suspension preparation
The A-9 bacterial strain slant strains that becomes ash is put into 4 ℃ of refrigerators, refrigerate and take out after 3-5 days, physiological saline 10-12ml with the bacterium of going out washes spore to come carefully, transfer in the aseptic triangular flask of 250ml that 30-40 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.Monospore strainer filtration with special use obtains monospore suspension then.Centrifugal collection monospore, resuspending are adjusted spore concentration 3 * 10 in above-mentioned stroke-physiological saline solution
7Individual/ml.
2.1.2 ultraviolet mutagenesis is handled
Above-mentioned monospore suspension is sub-packed in the aseptic little culture dish of 5 covers (in magnetic stirring bar is arranged), every ware 4-5ml.Draw 0.5ml bacterium liquid dilution back in addition and measure viable count, in contrast.Ultraviolet lamp power is 15w, and bacterium liquid is apart from 28 centimetres of fluorescent tubes, shines 20 seconds, 40 seconds, 60 seconds, 90 seconds and 120 seconds respectively, all operates in the darkroom and carry out under the ruddiness.Start magnetic stirrer during irradiation.
2.13 being coated with ware cultivates
Bacterium liquid after above-mentioned 5 kinds of UV processing is diluted to 10 respectively
-3, 10
-4, 10
-5, 10
-6Four kinds of concentration, every kind of concentration coating 5 cover malt meal inorganic salt agar plates (the prescription face that sees before is described, adding 1ml bacterium liquid in every cover is dull and stereotyped).Flat board after the coating is finished writing label, wraps with the went out black cloth of bacterium of multilayer, and in the sealed aluminum box of packing into, cultivates about 7 days in 27-28 ℃ of incubator.
2.1.4 sterilizing rate is measured
Measure contrast according to a conventional method abreast and, spend the sterilizing rate that calculates different irradiation dose UV treatment through the spore count alive in the liquid of bacterium everywhere of UV treatment.
2.1.5 choose single bacterium colony and shake the bottle screening
After treating the positive change of single bacterium colony ash, on the low slightly flat board of sterilizing rate, select the steamed bun shape bacterium colony that positive grey is dark, the back side is brown deeply, spore is plentiful, the slant medium of transferring again after the number of finishing.Through 27-28 ℃, carry out shake flask fermentation respectively after constant temperature culture to slant strains was grown well in 5-7 days.Measure tiring of Avrmectin in each bacterial strain fermentation liquor and be secreted into the ratio of the Avrmectin in the fermented supernatant fluid with the HPLC method.Through primary dcreening operation, multiple sieve and whole sieve, selected at last UV-F-106 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/L Tris of filtration sterilization and 0.1mol/L maleic acid balanced mix is that 8.0 damping fluid washes UV-F-106 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, the concentration to 10 of having adjusted spore
8Individual/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 Fructus Hordei Germinatus starch inorganic salt agar plate.Dilution-plate method separates single bacterium colony routinely.Select positive grey, back side brown is darker, the steamed bun shape bacterium colony switching inclined-plane that spore is plentiful.Treat that slant strains is grown and shake the bottle screening after good, choose and abamectin fermentedly tire higher and the also higher bacterial strain of the plain secretion rate of A Wei is made freeze-drying and guaranteed the Tibetan.
The Avrmectin ultimate production and the 5 all high strain A Fumai streptomycetes of secretion rate that are obtained by above method are: A Fumai streptomycete LAF-19, LAF-28, LAF-108, LAF-235 and LAF-307.
These bacterial strains can be secreted into the extracellular with its synthetic Avrmectin, the Avrmectin that secretion is come out is suspended in the upper strata of fermented liquid with the form of a kind of oil (wax) shape thing, the content of Avrmectin is about 20% in oil (wax) shape thing, and this is very beneficial for reducing the refinement cost of Avrmectin.
A Fumai streptomycete LAF-108 on September 28th, 2000 in typical culture collection center (Wuhan) preservation of specified depositary institution of State Intellectual Property Office China, preserving number is CCTCCNO:M200028.
Use bacterial strain of the present invention carry out in batches, semicontinuous or continuously ferment and can adopt the conventional substratum of producing Avrmectin, cultivate and fermentation condition can be conventional cultivation and fermentation condition.On the basis of existing batch fermentation processing unit, realize of the present invention in batches, semicontinuous or only continuously ferment that need carry out local flow improvement to equipment.
In the batch fermentation that uses above bacterial strain to carry out, after the fermentation ends upper strata oil (wax) shape thing is taken out, can obtain highly purified product with a small amount of organic solvent extracting again.Wherein the usage quantity of organic solvent only is about the 20-30% of existing batch fermentation technology.
Above bacterial strain also is fit to carry out semicontinuous or continuously ferments, promptly can realize continuing to flow the Ensure Liquid thing and the continuous continuous production mode of production of discharging oil (wax) shape thing, and realization or intermittent flow Ensure Liquid thing and or the semicontinuous mode of production of intermittently discharged oil (wax) shape thing.Semicontinuous or continuous ferment process uses in the production of some leavened prods, but the report that does not also use in Avrmectin is produced is being because the generation bacterium of generally adopting all is a nonsecreting type at present.Adopt bacterial strain of the present invention can reduce numerous and diverse preparation work in traditional batch fermentation technology, prolong the generated time of Avrmectin, reduce energy consumption, enhance productivity, reduce and refine cost.
Use bacterial strain of the present invention carry out in batches, semicontinuous or continuously ferment and can reduce the consumption of organic solvent significantly, so not only can reduce the refinement cost, but also help direct labor's healthy, labor safety and environment protection.
Can adopt following process carry out bacterial strain of the present invention in batches, semicontinuous or continuously ferment.
Batch fermentation
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.Guarantee that the discharging of oozy oil (wax) shape thing is convenient and the pipeline valve sterilization is convenient.
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 A Fumai streptomycetes if find varied bacteria growing.In 27-29 ℃ of thermostat container, cultivated 5-7 days, treat to collect after the spore maturation, be stored in the refrigerator and use for fermentation.
The seeding tank batch charging coefficient is 60-70%.The seed culture medium composition is: starch 2-4%, soybean cake powder 0.5-1.0%, 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.Steam sterilizing treats that substratum is cooled to the slant pore suspension of inoculation above-mentioned desinsection streptomycete in back about 28 ℃.Inoculum size is about 1 eggplant bottle of every 100L seed culture fluid slant strains, 1 test tube slant bacterial classification of perhaps every 3.25L substratum.At air flow 1: 0.5-1.0,26-28 ℃, 500-600 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 should 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 can be: starch 5-8%, active dry yeast powder 1.0-1.4%, Semen Maydis powder 1%, 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 0.004-0.006%, and add the mixing defoamer 0.01-0.03% contain bubble enemy and vegetables oil in advance, and be settled to volume requiredly with tap water, preceding pH6.8-7.0 disappears.Behind steam sterilizing, inoculate, inoculum size is 1-5%, 27-29 ℃ of jar temperature, air flow 1: 0.8-1.15 cultivated under the stirring velocity 250-350 rev/min of condition 9-10 days, rose to more than 7.5 to pH, the obvious self-dissolving of mycelia, when the B1a component of Avrmectin reaches peak-peak, open the discharge orifice valve, discharge float over the fermented liquid upper strata contain vegetable oil (wax) shape thing.Make to contain vegetable oil (wax) shape thing and directly enter extractor, with 2-4 times of volumes toluene (or ethyl acetate) extraction, decolorizing with activated carbon.The destainer reconcentration adds crystal seed at last and obtains the Avrmectin crystallization.
Semicontinuous fermentation
The same with above batch fermentation, 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.
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 5-8%, 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 0.2-0.3%, fire resistant alpha-diastase 0.004-0.006%, and add the mixing defoamer 0.01-0.03% of bubble enemy and vegetables oil, and be settled to volume requiredly with tap water, preceding pH 6.8-7.0 disappears.After eliminating bacterium, 130-150 ℃ of steam connection enter the fermentor tank inoculation.Inoculum size is 1-5%, 27-28 ℃ of jar temperature, and air flow 1: 1-1.1 5, stirring velocity 250-350 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 0.6%, reducing sugar begins feed supplement.The nutrient solution prescription that is replenished is: glucose 5-10%, vegetables oil (preferably soya-bean oil and peanut oil) 1-10%, bubble enemy 0.05-0.1%, cobalt chloride 4-6 μ g/ml (final concentration), Repone K 0.2-0.5%.Whether the microscopy mycelia aging of taking a sample since the 9th day every 8-12 hour.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 is once checked in sampling in per 12 hours, if the visible significantly oil of naked eyes (wax) shape thing is floating, or the HPLC method records Avrmectin B1a component concentration and is higher than 100 μ g/ml, then the short period of time can stop to stir and ventilation, open the above-mentioned discharge orifice valve of going up simultaneously, discharge and float over containing of fermented liquid upper strata of plain clear liquid.The amount of the fresh medium that at every turn is pressed into is equivalent to the 2%-8% of original fermented solution volume.
Should replenish fresh culture and suitably adjust fermentation condition by feed supplement, prolong above fermenting process as far as possible, improve the Avrmectin ultimate production.The plain liquid that contains of discharging from last discharge orifice directly enters extractor at every turn, carries out the extraction of secondary cross-current type with the toluene or the ethyl acetate of 2 to 4 times of volumes.Aqueous phase discarded concentrates toluene or ethyl acetate phase, adds the Avrmectin crystal seed, obtains the Avrmectin crystallization.
After some days, produce bacterial strain and all occur old and feeble self-dissolving inevitably, if the obvious old and feeble self-dissolving of mycelia then can be put jar.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 and feed supplement hole.Connect by corresponding pipeline valve between jar, concrete mode of connection is seen Figure of description 1.
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 A Fumai streptomycetes if find varied bacteria growing.In 27-29 ℃ of thermostat container, cultivated 5-7 days, treat to collect 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 can be 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 about 30 ℃.
The dilution range that adopts in the continuous fermentation process is 0.01-0.03 (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 25-26 ℃; The pH:1 jar is 6.4-6.8, and No. 2 jars (or No. 2 jars and No. 3 jars) are 6.2-6.5, and No. 3 jars (or No. 3 jars and No. 4 jars) are 6.5-6.8; 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 the stream that is in due course in jar is with nutriment such as maltose, glucose, vegetables oil, yeast extract, corn steep liquors.Jar in the end, i.e. 3 or No. 4 jar discharges, marker method can carry out with reference to semicontinuous fermentation.Also can get rid of sophisticated fermented liquid evenly incessantly.
Send out the plain fermented liquid that contains that to discharge and directly enter extractor.Extraction solvent is the secondary cross current solvent extraction with toluene or ethyl acetate, extraction mode.Go out the organic solvent phase by the disc centrifuge centrifugation, aqueous phase discarded and solid shape precipitation.Decolorizing with activated carbon, concentrating under reduced pressure (temperature is between 50-65 ℃) contains the organic solvent phase of Avrmectin, adds the Avrmectin crystal seed and carries out crystallization, can obtain the Avrmectin crystal.
After continuously fermenting some days, the same semicontinuous fermentation of treatment process after the catabiosis generally appears in the cell of production bacterium.
Below be embodiments of the invention, these embodiment are used for illustrating the present invention, but do not constitute any limitation of the invention.Embodiment 1 uses A Fumai streptomycete LAF-108 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 LAF-108 (being CCTCC NO:M200028) 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.
Prepare the 5L seeding tank, batch charging coefficient 65%.The seed culture medium composition is: starch 3%, soybean cake powder 0.8%, groundnut meal 0.9%, active dry yeast powder 0.5%, corn steep liquor 0.4%, cobalt chloride O.0008%, defoamer 0.03% 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 slant pore suspension of inoculation LAF-108 bacterial strain after 28 ℃.Inoculum size is 1 test tube slant seed of every jar of seed culture medium (3.25L).Air flow 1: 0.8,27 ℃, 550 rev/mins of agitation conditions were cultivated 24 hours down, and thicker to outward appearance, the microscopy mycelia is sturdy, multi-branched, under the no living contaminants situation culture transferring to 100L with in the fermentor tank of discharge orifice.
The fermentation cylinder for fermentation substratum is: starch 7%, 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 pH6.9 disappears.Inoculation after sterilizing, inoculum size is 3%, 28 ℃ of jar temperature, air flow 1: 0.9, stirring velocity was cultivated 9 days down for 300 rev/mins, and after fermentation 5 days, reducing sugar content is lower than at 0.6% o'clock in the fermented liquid, adds glucose by feed supplement jar stream and makes it to 1.4%.Mend sugar repeatedly.When pH rises to more than 7.5, the obvious self-dissolving of mycelia when the B1a component of Avrmectin reaches peak-peak, is opened the discharge orifice valve, discharge float over the fermented liquid upper strata contain vegetable oil (wax) shape thing (1181.0ml).The content of Avrmectin is 20% in oil (wax) shape thing.With the extraction of 3300ml toluene, decolorizing with activated carbon.The destainer reconcentration adds crystal seed at last and obtains Avrmectin crystallization 65.0 grams, wherein B1a component 58.5 grams.Embodiment 2 uses A Fumai streptomycete LAF-19 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 LAF-19 bacterial strain if find varied bacteria growing.In 29 ℃ 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 5L seeding tank, batch charging coefficient 65%.The seed culture medium composition is: starch 2%, and soybean cake powder 1.0%, groundnut meal 0.6%, active dry yeast powder 0.7%, corn steep liquor 0.3%, cobalt chloride 0.0003%, defoamer 0.02% 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 slant pore suspension of inoculation LAF-19 bacterial strain after 28 ℃.Inoculum size is test tube slant spore of every seed tank culture base (3.25L) inoculation.Air flow 1: 0.5,27 ℃, 550 rev/mins of agitation conditions were cultivated 36 hours down, and thicker to outward appearance, the microscopy mycelia is sturdy, multi-branched, under the no living contaminants situation culture transferring to 100L with in the fermentor tank of discharge orifice.
The fermentation cylinder for fermentation substratum is: starch 8%, active dry yeast powder 1.0%, Semen Maydis powder 1%, Repone K 0.6%, potassium primary phosphate 0.03%, cobalt chloride 5 μ g/ml (final concentration), sal epsom 0.03%, lime carbonate 0.2%, fire resistant alpha-diastase 0.006%.And add the mixing defoamer 0.01% that contains bubble enemy and vegetables oil in advance, and being settled to 70L with tap water, preceding pH7.0 disappears.Inoculation after sterilizing, inoculum size is 5%, 28 ℃ of jar temperature, air flow 1: 0.8 is cultivated under 300 rev/mins of conditions of stirring velocity, when reducing sugar content is lower than 0.6% in the fermented liquid, adds benefit sugar by feed supplement jar stream.Cultivated altogether 10 days, and rose to more than 7.5 to pH, the obvious self-dissolving of mycelia when the B1a component of Avrmectin reaches peak-peak, is opened the discharge orifice valve, discharge float over the fermented liquid upper strata contain vegetable oil (wax) shape thing (1127.0L).The content of Avrmectin is about 20% in oil (wax) shape thing.With the extraction of 3000ml toluene, decolorizing with activated carbon.The destainer reconcentration adds crystal seed at last and obtains Avrmectin crystallization 61.5 grams, wherein B1a component 55.3 grams.Embodiment 3 uses A Fumai streptomycete LAF-28 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 LAF-28 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 5L 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 slant pore suspension of inoculation LAF-28 bacterial strain after 28 ℃.Inoculum size is test tube slant spore of every seed tank culture base (3.25L) inoculation.Air flow 1: 1.0,27 ℃, 550 rev/mins of stir culture 12 hours, thicker to outward appearance, the microscopy mycelia is sturdy, multi-branched, under the no living contaminants situation culture transferring to 100L with in the fermentor tank of going up discharge orifice.
The fermentation cylinder for fermentation substratum is: starch 5%, active dry yeast powder 1.4%, Semen Maydis powder 1%, 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 0.004%.And add the mixing defoamer 0.03% that contains bubble enemy and vegetables oil in advance, and being settled to 70L with tap water, preceding pH6.8 disappears.Inoculation after sterilizing, inoculum size is 1%, 27 ℃ of jar temperature, air flow 1: 1.5 was cultivated under 300 rev/mins of conditions of stirring velocity 9 days, rose to more than 7.5 to pH, the obvious self-dissolving of mycelia, when the B1a component of Avrmectin reaches peak-peak, open the discharge orifice valve, discharge float over the fermented liquid upper strata contain vegetable oil (wax) shape thing (1058.0L).The content of Avrmectin is 21% in oil (wax) shape thing.With the extraction of 3000ml toluene, decolorizing with activated carbon.The destainer reconcentration adds crystal seed at last and obtains Avrmectin crystallization 60.6 grams, wherein B1a component 53.4 grams.Feed supplement method and main technologic parameters are seen embodiment 1.Embodiment 4,5,6,7 uses LAF-19, LAF-28, LAF-235 and LAF-307 bacterial strain to produce Avrmectin by batchwise
Adopt and embodiment 1 identical step, just LAF-108 is replaced with LAF-19, LAF-28, LAF-235 and LAF-307 bacterial strain respectively, the output of the Avrmectin that obtains at last is:
| Embodiment | Bacterial strain | Avrmectin (gram) | Avrmectin B1a (gram) |
| Embodiment 4 | ?LAF-19?? | ????61．5 | ????55．3 |
| Embodiment 5 | ?LAF-28 | ????60．6 | ????53．4 |
| Embodiment 6 | ?LAF-235 | ????59．1 | ????52．6 |
| Embodiment 7 | ?LAF-307 | ????56．3 | ????50．7 |
Adopt cultivation and the fermentation condition identical, change bacterial strain into the A9 bacterial strain with embodiment 1.Different is to put jar when the B1a of Avrmectin component reaches peak-peak.Filter press is collected mycelium, adjusts water content with light calcium carbonate and enters the tablets press granulation when moderate.With 70L acetone extraction Avrmectin.Vat liquor eliminates acetone through concentrating under reduced pressure, again with the extraction of 7000ml toluene, decolorizing with activated carbon.The destainer reconcentration adds crystal seed at last and obtains Avrmectin crystallization 57.5 grams, wherein B1a component 52.3 grams.
In the present embodiment, 100L ferment tank liquid is put tank volume 70L, can obtain wet mycelium 11.7Kg, and lixiviate spends acetone 70L, and acetone reclaims rate of loss more than 10%, counts 1 jar of loss acetone 7L.The usage quantity of toluene is also far above embodiment 1 in the present embodiment.As seen use organic solvent (acetone and the toluene) consumption of the technology of nonsecreting type bacterial strain to be far longer than the consumption of the technology of using the secretor type bacterial strain, the amount that the latter extracts required organic solvent only is 23.6% of the former aequum, according to present stage solvent calculation of price, adopt latter's solvent consumption amount of money only to be the former 10%.Embodiment 8 uses A Fumai streptomycete LAF-108 bacterial strain to press semi-continuous type and produces 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.
The process that slant strains is prepared and seed liquor prepares is with embodiment 1.Fermention medium (starch 7%, active dry yeast powder 1.2%, the Semen Maydis powder 1.0% of preparation semicontinuous fermentation, soybean cake powder 1.2%, corn steep liquor 0.6%, 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%), press the mixing defoamer that 0.02% of culture volume adds bubble enemy and vegetables oil, be settled to 70L with tap water, preceding pH6.9 disappears.Enter the fermentor tank inoculation after 140 ℃ of steam connection are eliminated bacterium, inoculum size is 3%, 28 ℃ of jar temperature, and air flow 1: 1.1,300 rev/mins of condition bottom fermentations of stirring velocity are 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 6 hours.When being lower than 0.6%, reducing sugar begins feed supplement.The nutrient solution prescription that is replenished is: glucose 8%, vegetables oil (soya-bean oil) 6%, bubble enemy 0.08%, cobalt chloride 5 μ g/ml (final concentration), Repone K 0.4%.Whether the microscopy mycelia aging of taking a sample since the 9th day per 10 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, sampling in per 12 hours once, check fermented liquid supernatant liquid part Avrmectin B1a component concentration, when the HPLC method records Avrmectin B1a component concentration and is higher than 100 μ g/ml, short period of time stops to stir and ventilation, open above-mentioned discharge orifice valve simultaneously, discharge float over the fermented liquid upper strata contain vegetable oil (wax) shape thing, the about altogether 3000ml of this oil (wax) shape thing and water.Therefrom separate fuel-displaced (wax) shape thing 255ml, wherein the content of Avrmectin is 18.5%.Then, add fresh medium, repeat above-mentioned fermenting process 11 times, obtain to contain vegetable oil (wax) shape thing 2780ml altogether.Obvious old and feeble self-dissolving appears in mycelia after 18 days, puts jar this moment.The amount that at every turn covers fresh medium is equivalent to 4.5% of original fermented solution volume approximately.Make vegetable oil (wax) the shape thing that contains of at every turn discharging directly enter extractor, carry out the secondary cross-current type with the toluene of 2.5 times of volumes and extract from discharge orifice.Aqueous phase discarded concentrates the toluene phase, adds Avrmectin B1a crystal seed, obtains the Avrmectin crystallization, obtains Avrmectin 140.4 grams altogether, wherein obtains Avrmectin B1a component 123.6 grams.
Press the technology of present embodiment, the 100L fermentor tank obtained 140.4 gram Avrmectins in 18 days, obtained 65.0 grams than 9 days of embodiment 1 and had improved working efficiency.The consumption ratio of organic solvent and embodiment 1 are similar.Embodiment 9,10,11,12 uses A Fumai streptomycete LAF-19, LAF-28, LAF-235 and LAF-307 bacterial strain to press semi-continuous type and produces Avrmectin
Adopt and embodiment 8 identical steps, just LAF-108 is replaced with LAF-19, LAF-28, LAF-235 and LAF-307 bacterial strain respectively, the output of the Avrmectin that obtains at last is:
| Embodiment | Bacterial strain | Avrmectin (gram) | Avrmectin B1a (gram) |
| Embodiment 9 | ?LAF-19 | ????133．5 | ????121．4 |
| Embodiment 10 | ?LAF-28 | ????131．2 | ????120．7 |
| Embodiment 11 | LAF-235 | ????129．1 | ????117．5 |
| Embodiment 12 | ?LAF-307 | ????121．0 | ????107．9 |
According to foregoing malt meal starch inorganic salt agar slant culture-medium formulation eggplant bottle 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 7 days, treat to collect after the spore maturation, be stored in 4 ℃ of refrigerators.Carry out species test with shaking bottle mode before last jar, be used for fermentative production after species test is qualified.
Fermentor tank is transformed with embodiment 8, the right cylinder position No. 2 and No. 3 fermentor tank the same sides, and the about tank body total height of height 70% place opens a discharge orifice, and establishes material-feeding port on No. 2 and No. 3 jars, at last discharge orifice and material-feeding port corresponding pipeline valve is installed.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 8).Fermention medium is by continuous sterilization system sterilization, is stored in the fermention medium basin after cooling the temperature to about 30 ℃.
The dilution range that adopts in this continuous fermentation process is 0.02 (l/h), and the series can number is 3, and the working volume of each jar is 100L, and volumetric ratio is 1: 1: 1.Temperature: No. 1 jar and No. 2 jars are 28 ℃, and No. 3 jar is 26 ℃; The pH:1 jar is that 6.5, No. 3 jars of 6.8, No. 2 jars are 6.8; Air quantity: No. 1 jar is 1: 1.5, and No. 2 jar is 1: 1.2, and No. 3 jar is 1: 1.0.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, and stirring velocity is 300 rev/mins.The parameter that provides with reference to embodiment 8 when the needs feed supplement in jar stream with nutriment such as maltose, glucose, vegetables oil, yeast extract, Semen Maydis powder.Jar in the end, i.e. No. 3 jar discharges, marker method can carry out with reference to embodiment 8, also can discharge sophisticated fermented liquid at the uniform velocity incessantly.The content of Avrmectin is 19% in oil (wax) shape thing.After so continuously fermenting 17 days, catabiosis generally appears in the cell of producing bacterium, stops fermentation and puts jar.
Vegetable oil (wax) the shape thing that contains of discharge is separated through disc centrifuge with the mixture of water, and aqueous phase discarded (heavy-fluid) keeps light liquid.Make light liquid directly enter extractor.Extraction solvent is the toluene of 3 times of volumes, and the extraction mode is the secondary cross current solvent extraction.Isolate the toluene phase by tubular-bowl centrifuge, discard solid shape precipitation.Decolorizing with activated carbon, concentrating under reduced pressure (temperature≤60 ℃) contains the toluene phase of Avrmectin, adds Avrmectin B1a crystal seed and carries out crystallization, obtains the Avrmectin crystal.Obtain Avrmectin 492.0 grams altogether, wherein Avrmectin B1a component 433.0 grams.
Press the technology of present embodiment, 3 jars obtained 492.0 gram Avrmectins in 17 days, obtained 65.0 grams in 9 days than 1 jar of embodiment 1 and had further improved working efficiency.The consumption ratio of organic solvent and embodiment 1 are similar.Embodiment 14,15,16,17 uses A Fumai streptomycete LAF-19, LAF-28, LAF-235 and LAF-307 bacterial strain to press continuous fermentation method and produces Avrmectin
Adopt and embodiment 13 identical steps, just LAF-108 is replaced with LAF-19, LAF-28, LAF-235 and LAF-307 bacterial strain respectively, the output of the Avrmectin that obtains at last is:
| Embodiment | Bacterial strain | Avrmectin (gram) | Avrmectin B1a (gram) |
| Embodiment 9 | LAF-19 | 481.6 | 428.5 |
| Embodiment 10 | LAF-28 | 471.3 | 424.2 |
| Embodiment 11 | LAF-235 | 468.0 | 416.5 |
| Embodiment 12 | LAF-307 | 448.8 | 407.9 |
Description of drawings
Fig. 1 is the synoptic diagram of fermentor tank mode of connection and Flow of Goods and Materials in the continuous fermentation process of the present invention.Wherein 1, connect 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, disc centrifuge
12, continuous sterilisation tower
13, keep jar
14, plate-type heat exchanger
15, temperature is reduced to the no bacteria fermentation culture medium about 30 ℃
16, feed supplement jar
17, the mixed solution that contains avermitilis strain vegetable oil (wax) shape thing and fermented liquid from giving off
18, connect extraction process
Claims (9)
1. the novel process of a fermentative Production Avrmectin is characterized in that using during the fermentation can producing Avrmectin and Avrmectin being secreted into the outer microorganism of somatic cells.
2. technology as claimed in claim 1, wherein said microorganism are A Fumai streptomycete (Streptomyces avermitilis).
3. technology as claimed in claim 2, wherein said microorganism are A Fumai streptomycete CCTCCNO:M200028.
4. as the technology of claim 1 or 2, wherein said fermentation is a batch fermentation.
5. as the technology of claim 1 or 2, wherein said fermentation is a semicontinuous fermentation.
6. as the technology of claim 1 or 2, wherein said fermentation is to continuously ferment.
7. can produce Avrmectin and Avrmectin is secreted into the outer A Fumai streptomycete of somatic cells.
8. A Fumai streptomycete CCTCC NO:M200028.
9. Avrmectin be can produce and varient or the mutant of the outer A Fumai streptomycete CCTCC NO:M200028 of somatic cells Avrmectin are secreted into.
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| CNB001302337A CN1163617C (en) | 2000-10-30 | 2000-10-30 | Process for preparing evericin by fermentation method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100368553C (en) * | 2004-10-09 | 2008-02-13 | 武汉绿世纪生物工程有限责任公司 | Production bacterial strain of abamectin with high yield and high secreation rate and new method of extracting AVM |
| CN100424090C (en) * | 2006-09-27 | 2008-10-08 | 河北瑞通美邦工程有限公司 | Extraction technology of avermectin |
| CN101560535B (en) * | 2009-05-26 | 2012-05-23 | 浙江升华拜克生物股份有限公司 | Process for producing abamectin by feeding glucose fermentation based on metabolizing parameters OUR |
| CN101659970B (en) * | 2009-07-30 | 2012-07-04 | 浙江升华拜克生物股份有限公司 | Method for circularly treating avermectins waste ferment water and pleurin waste ferment water |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102217591A (en) * | 2011-04-26 | 2011-10-19 | 张福志 | Environmentally-friendly abamectin ointment production process |
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2000
- 2000-10-30 CN CNB001302337A patent/CN1163617C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100368553C (en) * | 2004-10-09 | 2008-02-13 | 武汉绿世纪生物工程有限责任公司 | Production bacterial strain of abamectin with high yield and high secreation rate and new method of extracting AVM |
| CN100424090C (en) * | 2006-09-27 | 2008-10-08 | 河北瑞通美邦工程有限公司 | Extraction technology of avermectin |
| CN101560535B (en) * | 2009-05-26 | 2012-05-23 | 浙江升华拜克生物股份有限公司 | Process for producing abamectin by feeding glucose fermentation based on metabolizing parameters OUR |
| CN101659970B (en) * | 2009-07-30 | 2012-07-04 | 浙江升华拜克生物股份有限公司 | Method for circularly treating avermectins waste ferment water and pleurin waste ferment water |
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