CN1231577C - Thuringinensin bacillus solid pelletizing and cultivating method - Google Patents
Thuringinensin bacillus solid pelletizing and cultivating method Download PDFInfo
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- CN1231577C CN1231577C CN 200310104078 CN200310104078A CN1231577C CN 1231577 C CN1231577 C CN 1231577C CN 200310104078 CN200310104078 CN 200310104078 CN 200310104078 A CN200310104078 A CN 200310104078A CN 1231577 C CN1231577 C CN 1231577C
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Abstract
The present invention relates to a cultural method for Bacillus thuringiensis solid granulating, which is characterized in that the method comprises that (1) a material of a solid culture medium is mechanically pulverized to reach pulverizing fineness of 200 to 400 meshes; (2) the pulverized material of the solid culture medium is added in water to humidified and uniformly stirred, and water quantity is 60 to 80% of the total weight of the material of the solid culture medium; (3) the humidified material of the solid culture medium is shaped to form granules, and the diameter of the position of the maximum cross section of each shaped granule is from 2 to 10mm; (4) the shaped granules are sterilized; (5) strain liquid is added in the shaped granules to be fermented and cultured. The cultural method for Bacillus thuringiensis solid granulating greatly shortens a fermentation period, conspicuously improves the yield of the effective ingredient of a product, and increases producible varieties; the present invention has the advantages of low labor intensity, low production cost and smaller production energy consumption, and the toxicity potency of the fermentation product reaches 14000 to 16000 iu/mg.
Description
Technical field
The present invention relates to a kind of manufacture method of microbial pesticide, especially a kind ofly be used to prepare microbial pesticide---bacillus thuringiensis cultural method.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis) is called for short B.T, is the microbial pesticide of present worldwide production maximum.It is widely used in control Agricultural pests, forest and fruit tree pest insect, storage insect and medical insect, and has obtained remarkable economic efficiency, social benefit and ecological benefits.Particularly under the situation of being badly in need of the development non-polluted farm product, use bacillus thuringiensis can significantly reduce the residual of chemical pesticide, stop because of using person poultry poisoning's phenomenon that chemical pesticide causes.
In the prior art, one class is to adopt the deep fermentation technology to cultivate bacillus thuringiensis (is 91105123.6 Chinese invention patent application disclosed " kind of plant with the production method of bacterium for killing insect " etc. as application number), these class methods need main equipments such as fermentor tank, air filtering system, plate-and-frame filter press, concentration tank, and complex process, investment are greatly, cost is high, power consumption is big; Another kind of employing solid-fermented technique comprises tray formula, box and trough type fermentation device cultivation bacillus thuringiensis.The solid-fermented technique technology of this quasi-tradition is simple, less investment but yield poorly.Because bacillus thuringiensis belongs to aerobic microbiological, the ventilation property difference will suppress normal growth and the metabolism of bacillus thuringiensis.Therefore, usually past culture base-material is (as wheat bran, rice bran, Semen Maydis powder, soybean cake powder, groundnut meal, cottonseed meal etc.) add raising agent in (as husk, sawdust, corn cob, sorghum stalks etc. are rich in coarse-fibred material) improve its ventilation property, but the fermentation to bacillus thuringiensis causes following adverse consequences with aftertreatment (making the process of different formulations): the 1. needed moisture of bacillus thuringiensis growing multiplication, air and nutritive substance come from the culture base-material solid particles surface, the adding of raising agent, having significantly reduced in the unit volume to reduce fermentation efficiency by the effective surface area that bacillus thuringiensis utilized; 2. the bacillus thuringiensis plain enzyme of eccrine fiber hardly, the raising agent of interpolation can not be utilized, and has both wasted raw material, makes again that the bacillus thuringiensis growing multiplication is slack-off, the production cycle prolongs, the toxicity evaluation of product is not high; 3. raising agent is difficult to separate and pulverize in fermented product, causes the performance (as fineness, stability, suspensibility etc.) of finished product not reach the correlated quality standard, seems more outstanding in the production of bacillus thuringiensis wettable powder.Therefore, the method for above-mentioned existing cultivation bacillus thuringiensis awaits improving.
Summary of the invention
The objective of the invention is the method for above-mentioned employing solid-fermented technique cultivation bacillus thuringiensis is improved, a kind of fermentation period that shortens is provided, reduce production energy consumption, increase the bacillus thuringiensis solid granulation cultural method of equipment capacity and raising product toxicity evaluation.
Bacillus thuringiensis solid granulation cultural method of the present invention is to carry out according to the following steps: 1., the solid culture based raw material that is used for the bacillus thuringiensis solid culture is carried out mechanical disintegration, smashing fineness is at 200～400 orders; 2., add water in the solid culture based raw material after pulverizing and make it humidifying and stir, amount of water is 60～80% of a solid culture based raw material gross weight; 3., be particle with the solid medium material forming of humidifying, the diameter at the position, maximum cross section of institute's shaped granule is 2～10mm; 4., the particle of moulding was sterilized 30～45 minutes down for 121～135 ℃ in pressure 0.1～0.15MPa, temperature; 5., make the shaped granule naturally cooling after the sterilising treatment, when its temperature is reduced to 28～30 ℃, the strain liquid of bacillus thuringiensis inserted carries out fermentation culture in this shaped granule, the fermentation culture temperature is that 27～37 ℃, humidity are 90～95%; When gemma, parasporal crystal appearance 40～60% in the moulding particle stop cultivating when dissociating.
Above-mentioned steps 1. in, the solid culture based raw material that is adopted can be the conventional raw material in the existing solid fermentation cultural method, as wheat bran, rice bran, Semen Maydis powder, soybean cake powder, groundnut meal, cottonseed meal etc.Simultaneously, each component in the solid culture based raw material is all prepared in the ratio of original processing requirement.That is to say how to determine the ratio of the raw material and the each component thereof of solid medium, all is not the inventive method problem to be solved.The step of the inventive method 1. in, only need directly the conventional raw material of preparing by existing solid culture method (promptly by existing composition of raw materials) to be implemented to pulverize, the fineness that makes it to reach certain gets final product.Mechanical disintegration to the solid culture based raw material can adopt common micronizing machinery to carry out.Step 2. in, institute's amount of water makes it to have the plasticity of extrusion molding can make solid culture base-material humidifying in the solid culture based raw material, and the particle that obtains after the moulding is indeformable, adhesion, smooth surface are as the criterion.Where necessary, other trace ingredients can also be dissolved in the water as yeast powder, sal epsom, sodium hydrogen phosphate, ferrous sulfate, zinc sulfate, lime carbonate etc., add in the solid culture based raw material that has stirred again.Step 3. in, can adopt existing extrusion formation equipment to realize granulation, prepared shaped granule can be the particle of cylindric, spherical shape, ellipsoid shape or similar spheroid-like.When moulding particle when being cylindric, its maximum cross section is the circular cross sections of cylinder, but not the length of cylinder; When the moulding particle was spherical shape, the diameter of its maximum cross section was the sphere diameter of ball; When the moulding particle was the shape of ellipsoid shape or similar ellipsoid, its maximum cross section diameter was the minor axis length of ellipsoid, and the long axis length of non-spheroid.Answer smooth mellow and full, no burr, do not ftracture in made particulate surface.With regard to step 4., employing is the conventional sterilising method that existing solid-fermented technique is cultivated bacillus thuringiensis.Step 5. in, gemma, parasporal crystal occur 40～60% and stop cultivating when free in detecting shaped granule, and do not consider the omnidistance time of fermentation culture.This is because when adopting different solid culture based raw materials or bacterial classification, the omnidistance time of its fermentation culture, bigger difference might take place.In addition, the strain liquid that inserts in this shaped granule can be the strain liquid that is made by the bacillus thuringiensis inclined-plane, also can be the strain liquid that is made by three grades of seeding tanks, the culture condition of applied bacillus thuringiensis be in fact identical with solid fermentation culture condition of the prior art.After cultivating end, can carry out aftertreatment to cultivating products therefrom by existing processing requirement, and then make finished products such as water suspending agent, wettable powder.
1., adopt solid granulation culture technique the invention has the beneficial effects as follows:, under culture condition, form hole between particle and the particle, can keep competent oxygen and ventilate smoothly, guarantee bacillus thuringiensis demand to nutrition and ventilation property in the solid culture process.Avoid raising agent to bring adverse consequences for fermentation and aftertreatment effectively.Especially be suitable for the large-scale industrial production of bacillus thuringiensis solid fermentation.2., traditional solid fermentation will carry out spice, very easily microbiological contamination immediately after inoculation.Adopt after the solid granulation culture technique, because particulate smooth surface and hole effect, can save this operation of spice of inoculation back, i.e. culture base-material material surface sprinkling strain liquid of only being piled into by shaped granule, strain liquid just can be deposited on each shaped granule naturally, and can uniform distribution, reduced the probability and the labour intensity of microbial contamination in the bacillus thuringiensis production process.3., adopt after the solid granulation culture technique, make bacillus thuringiensis only grow into thick lawn at particle surface, granule interior does not then form.In postprocessing working procedures, the easy and particle separation of the lawn on surface not only makes the recovery rate of effective constituent improve, and makes the bacillus thuringiensis product of high toxicity evaluation easily.4., solid granulation culture technique can combine with existing any bacillus thuringiensis solid fermentation device, when improving the product toxicity evaluation, can also shorten fermentation period, cuts down the consumption of energy, and increases the throughput of equipment.
In the present invention, in order to allow shaped granule have comparatively slick surface, and between shaped granule, form enough holes, can allow the 3. prepared shaped granule of step have the particle of curved surfaces for the surface.
Content of the present invention further illustrates with the following Examples, but content of the present invention is not limited only to content related among the embodiment.
Embodiment
Bacillus thuringiensis solid granulation cultural method in the present embodiment carries out (unit of following related each weight part is kilogram) according to the following steps:
1., the solid culture based raw material that is used for the bacillus thuringiensis solid culture is carried out mechanical disintegration, smashing fineness is 300 orders.In the present embodiment, adopted existing solid medium composition of raw materials, pulverized solid culture based raw material is wheat bran, soybean cake powder and Semen Maydis powder, and three's weight part ratio is 82.2 to 2 to 2.Mix according to the above ratio after the pulverizing.
2., add water in the solid culture based raw material after pulverizing and make it humidifying and stir, amount of water is 70% of a solid culture based raw material gross weight.In the present embodiment, when in the solid culture based raw material, adding water, weight part is respectively 1.5,0.03,0.3 trace ingredients yeast powder, sal epsom and lime carbonate to be added water and stirs, unslaked lime (weight part 1.25) is added the solution that water becomes behind the lime aqueous solution with yeast powder etc. is made separately mix, add again in the above-mentioned pulverized solid culture based raw material.In addition, be that 4.5 fish meal added water boil 35 minutes with weight part, be that sterilization was stand-by separately to make nutritive medium after 2 glucose mixed with weight part.
3., the solid medium material forming with humidifying is a particle.In the present embodiment, utilize extrusion equipment, the solid culture base-material that mixes is squeezed into cylindrical pellet, the diameter at its position, particulate maximum cross section (being the cross-sectional diameter of cylinder) is 4mm, and particle length is at 3cm.Above-mentioned shaped granule has the particle of curved surfaces for the surface.
4., particle (being the particulate state culture base-material) the immigration high-temperature sterilization pot of moulding is sterilized.Sterilization pressure is 0.1MPa, and temperature was sterilized 40 minutes at 121 ℃, treats that the temperature of culture base-material is reduced to 58 ℃, and pressure is to move in the culturing room in 0 o'clock.
5., make the shaped granule naturally cooling after the sterilising treatment, when its temperature is reduced to 29 ℃, the strain liquid of bacillus thuringiensis inserted carries out fermentation culture in this shaped granule, the fermentation culture temperature is that 27～37 ℃, humidity are 90～95%; When gemma, parasporal crystal appearance 40～60% in the moulding particle stop cultivating when dissociating.Detailed process is:
When the temperature of being piled up the culture base-material that forms by shaped granule is reduced to 29 ℃, the strain liquid of bacillus thuringiensis (strain number: HBNK-1) mix with the nutritive medium of half amount, the surface that sprays into culture base-material (need not the stir culture base-material, strain liquid just can be in intergranular hole natural subsidence, and can be evenly distributed on the particle).Cultivate preceding 1～12 hour, the nourishing body of bacillus thuringiensis was in logarithmic phase.Be cultured to 11～12 hours, nourishing body is in the logarithm peak phase.This moment, the nutritive medium with second half amount of remainder sprayed into the culture base-material surface, the extra-nutrition source.The temperature in this period is controlled at 28～30 ℃.Incubation time was at 13～25 hours, and this moment, the nourishing body of bacillus thuringiensis was in stationary phase, and the temperature in this period is controlled at 30～32 ℃.After 26 hours, the nourishing body of bacillus thuringiensis is in gemma and free stage of parasporal crystal, and the temperature of this moment is controlled at 34～36 ℃, and is aging to promote it.Incubation time to 32～34 hour have 50% gemma and parasporal crystal free, so stop cultivation.The whole fermentation process controlling moisture is 90～95%.If form the lawn of a thick layer of bacillus thuringiensis (with culture base-material convection drying, pulverizing after, measure its toxicity evaluation 14000～16000IU/mg) at particle surface this moment.
In the present embodiment, after above-mentioned cultivation finishes, carried out aftertreatment by existing processing requirement to cultivating products therefrom, its treatment process is:
A, will cultivate the solid culture base-material that finishes and pour the sterilized water that adds 1.8 times in the stirred pot into, mechanical stirring 30 minutes, be grown in the bacillus thuringiensis lawn on culture base-material surface, by the mutual friction between the ganoid particle, be washed into and become the bacterium of bacillus thuringiensis liquid in the water, and culture base-material can moltenly not scatter at short notice, very easily realizes separating of bacillus thuringiensis and solid culture base-material.
B, the mixed solution after will stirring carry out natural subsidence by the vibratory screening apparatus of 2mm, and granular culture base-material is filtered off, and carries out fine filtering by whizzer after the filtrate coarse filtration, and filtrate is again by behind the 200 order filter clothes, and packing into holds the basin of bacterium liquid for the first time.Filter residue is added sterilized water again, and repeated washing once again.The bacterium liquid that the obtains basin of bacterium liquid for the second time of packing into.Through 3 after backwashing, the recovery rate of effective ingredient is 93%.
C, last consignment of time is produced the first time bacterium liquid as water lotion, join in the culture base-material of next batch, repeat aforesaid operations and wash.Can obtain the bacterium liquid of the bacillus thuringiensis of high toxicity evaluation.
D, the bacterium liquid that classification is obtained directly prepare the water suspending agent product of 2000IU/ μ l (salable product), 4000IU/ μ l (first grade), 8000IU/ μ l (top grade product).
The bacterium liquid of E, the high toxicity evaluation that will obtain through concentrating, dry, can prepare the former powder of bacillus thuringiensis of 40000IU/mg (salable product), 50000IU/mg (first grade).Also can be with the wettable powder product of the solid culture base-material convection drying after the fermentation ends, the preparation of pulverizing back 8000IU/mg (salable product), 16000IU/mg (first grade).
In order to further specify the technique effect of the inventive method, below special providing adopt existing solid fermentation method to cultivate the example of bacillus thuringiensis, i.e. reference examples:
The composition of raw materials of reference examples: with the prescription of the above embodiment of the present invention.
Its production process is:
The first step: raw material (except fish meal, the glucose) is mixed in proportion, add 70～80% of Total Water and mix.Fish meal adds water boil 30-40 minute, and sterilization is stand-by separately with making nutritive medium after glucose mixes.
Second step: the solid culture base-material for preparing is moved into the high-temperature sterilization pot sterilize.Sterilization pressure is 0.1MPa, and temperature was sterilized 40 minutes at 121 ℃, treats that the temperature of culture base-material is reduced to 58 ℃, and pressure is to move in the culturing room in 0 o'clock.
The 3rd step: when the temperature of culture base-material is reduced to 29 ℃, the seed liquor of bacillus thuringiensis (strain number: HBNK-1) mix, insert in the sterilized solid culture base-material, uniform mixing, and once every 6 hours stirrings with nutritive medium.1～17 hour, the nourishing body of bacillus thuringiensis was in logarithmic phase, and the temperature in this period is controlled at 28～30 ℃.Incubation time was at 18～32 hours, and the nourishing body of bacillus thuringiensis is in stationary phase, and the temperature in this period is controlled at 30～32 ℃.33～50 hours, the temperature in this period was controlled at 32～34 ℃.The whole fermentation process controlling moisture is 90～95%.Gemma as 50% and parasporal crystal are free, just stop cultivating, and this moment, fermentation period was about 50 hours.And the surperficial naked eyes of culture base-material are not seen thick lawn.After culture base-material convection drying, pulverizing, measure its toxicity evaluation at 8000～10000IU/mg.
Now the basic condition with the embodiment of the invention (solid granulation cultivation) and reference examples (conventional solid culture) fermentation compares, and is listed as follows:
The solid granulation is cultivated and the basic situation synopsis of conventional solid culture
| Training method | Bacterial strain | Composition of raw materials | The fermentation container type | Intermediate controlled | Fermentation period | The culture base-material toxicity evaluation | Yield of effective components | The production kind | Labour intensity | Production cost | Production energy consumption |
| The solid granulation is cultivated | HBNK-1 | Referring to embodiment | Fermenter | Need add one time of nutrition liquid | 32～34 hours | 14000～16000 IU/mg | Wettable powder 100%, water suspending agent 90～95% | More relatively | Less relatively | Low | Less |
| Conventional solid culture | HBNK-1 | Referring to reference examples | Fermenter | Inoculation needs spice, at interval 6 hours stirrings | 48～50 hours | 8000～10000 IU/mg | Wettable powder 100%, water suspending agent 50～60% | Less relatively | Relatively large | High | Bigger |
From above synopsis as can be seen, compare with class methods with existing, adopt the fermentation period of bacillus thuringiensis solid granulation cultural method of the present invention to shorten dramatically, the toxicity evaluation of product and the recovery rate of effective ingredient significantly improve, producible kind increases, and labour intensity reduces, and production cost reduces, production energy consumption is also less, thereby has high using value.
Claims (3)
1, a kind of bacillus thuringiensis (Bacillus thuringiensis) solid granulation cultural method is characterized in that carrying out according to the following steps:
1., the solid culture based raw material that is used for the bacillus thuringiensis solid culture is carried out mechanical disintegration, smashing fineness is at 200～400 orders;
2., add water in the solid culture based raw material after pulverizing and make it humidifying and stir, amount of water is 60～80% of a solid culture based raw material gross weight;
3., be particle with the solid medium material forming of humidifying, the diameter at the position, maximum cross section of institute's shaped granule is 2～10mm;
4., the particle of moulding was sterilized 30～45 minutes down for 121～135 ℃ in pressure 0.1～0.15MPa, temperature;
5., make the shaped granule naturally cooling after the sterilising treatment, when its temperature is reduced to 28～30 ℃, the strain liquid of bacillus thuringiensis inserted carries out fermentation culture in this shaped granule, the fermentation culture temperature is that 27～37 ℃, humidity are 90～95%; When gemma, parasporal crystal appearance 40～60% in the moulding particle stop cultivating when dissociating.
2, the method for claim 1, it is characterized in that step 3. in, prepared shaped granule has the particle of curved surfaces for the surface.
3, method as claimed in claim 1 or 2, it is characterized in that step 5. in, by to the culture base-material surface sprinkling strain liquid of being piled into, finish strain liquid with bacillus thuringiensis and insert operation in the shaped granule by shaped granule.
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| CN 200310104078 CN1231577C (en) | 2003-12-22 | 2003-12-22 | Thuringinensin bacillus solid pelletizing and cultivating method |
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| CN 200310104078 CN1231577C (en) | 2003-12-22 | 2003-12-22 | Thuringinensin bacillus solid pelletizing and cultivating method |
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| CN1231577C true CN1231577C (en) | 2005-12-14 |
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| CN103160437A (en) * | 2011-12-15 | 2013-06-19 | 青岛高科技工业园海博生物技术有限公司 | Method for producing microbial granular culture medium |
| CN105165913A (en) * | 2015-10-22 | 2015-12-23 | 苏州科大微龙农业科技有限公司 | Sophocarpidine compound prevention and treatment agent for preventing and treating trialeurodes vaporariorum |
| CN109082385B (en) * | 2018-08-22 | 2021-09-14 | 四川省农业科学院土壤肥料研究所 | Preparation method of morchella liquid strain |
| CN109593680B (en) * | 2018-12-26 | 2022-02-22 | 武汉科诺生物科技股份有限公司 | Bacillus thuringiensis liquid fermentation medium and bacterial powder and oil suspension agent thereof |
| CN114456998A (en) * | 2022-03-23 | 2022-05-10 | 湖北天惠生物科技有限公司 | Production process of easily-crushed solid culture medium for bacillus thuringiensis biopesticide |
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