CN1459232A - Method for prepn. of bio-pesticide using new type engineering fungi (or bacteria) - Google Patents
Method for prepn. of bio-pesticide using new type engineering fungi (or bacteria) Download PDFInfo
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- CN1459232A CN1459232A CN 02120513 CN02120513A CN1459232A CN 1459232 A CN1459232 A CN 1459232A CN 02120513 CN02120513 CN 02120513 CN 02120513 A CN02120513 A CN 02120513A CN 1459232 A CN1459232 A CN 1459232A
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- engineering bacteria
- biopesticide
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Abstract
A process for preparing an agricultural biochemical features that a novel engineering bacterium TS16 which is the Bacillus thuring iensis as production strain obtained by artificially mutagenizing its wild bacterium strain, and the invented fermenting culture medium and technological flow path are used. Its advantages are high E/C ratio, low cost, high performance and no harm to domestic animals and people.
Description
The present invention relates to a kind of preparation method of biopesticide, particularly a kind of method of utilizing the novel engineering bacteria of bacillus thuringiensis,Bt to prepare biopesticide.
Since 1938 since first bacillus thuringiensis,Bt commodity preparation Sporeine is born in France, thuricade-1 has been obtained great success on producing.The scientist of China isolates bacillus thuringiensis strains in nineteen fifty-five from the commodity preparation of introducing, go out the bacterium powder in prepared in laboratory and be used for the control of maize snout moth's larva.Today, the production of thuricade-1 be applied in China and obtained remarkable achievement.Because that thuricade-1 has is free from environmental pollution, nontoxic to people and animals, do not injure non-target organism, and cost low, be easy to many advantages such as production, thereby, established its leading position in biological control of insect pests.
In recent years, in the research of artificial constructed engineering bacteria, it is found that by means of physics, chemical method and technique for gene engineering seed selection or make up new bacterial strain, is the shortcut that the nature remodeling bacterial strain obtains efficient Biotrol BTV.Bhattacharya (2000) obtains high virulence bacillus thuringiensis,Bt mutant strain with nitrosoguanidine (NTG) mutagenic treatment; Britain and The former Russian scholar once Applied Physics method mutagenic and breeding arrive efficient bacterial strain; The company of the U.S. obtains the bacterial strain higher 2 times than starting strain virulence after some plasmid of HD-1 is eliminated, and the conduction technology handles it, has obtained the virulence bacterial strain higher 7.5 times than starting strain; It is the microorganism insecticide of producing bacterial strain that Ecogen company has just registered with genetic engineering bacterium EG7673 in nineteen ninety-five.At present, though external had very big development in the research of bacillus thuringiensis,Bt, really the artificial constructed engineering bacteria of using on producing is also few, in China then still less.
China is also delivering many articles aspect the artificial constructed engineering bacteria, reported " structure of disinsection prophylaxis gene engineering bacillus subtilis " " biotechnology journal " in April, 1999 (15 2 phases of volume); Reported " Zhongshan University's journal " in July, 1999 (38 4 phases of volume) " utilizing transposons Tn917 to make up desinsection Bt engineering bacteria "; Reported " desinsection genetic engineering pseudomonas fluorescens IPP202 part biological is learned characteristic " " microorganism journal " February calendar year 2001 (41 1 phase of volume).Above article has been introduced China and has been adopted technique for gene engineering to make up the laboratory test situation of engineering bacteria.
Conventional biopesticide preparation method, its production technology is: sand pipe bacterial classification → slant activation → shake bottle investigation → seed tank culture → fermentation tank culture → filtering and concentrating → absorption → atomized drying → quality inspection → packing → finished product to put in storage.
Purpose of the present invention is to handle by the induced mutations to wild strain, obtains the novel engineering bacteria of bacillus thuringiensis,Bt efficient, wide spectrum, utilizes novel engineering bacteria to put into production, for agricultural production increases a kind of new biopesticide.
The method of utilizing novel engineering bacteria to prepare biopesticide of the present invention, it is the wild strain that utilizes from the occurring in nature screening and separating, handle novel engineering bacteria TS16 (the Bacillus thuringiensis of the efficient bacillus thuringiensis,Bt of acquisition through induced mutations, delivered the China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation that is positioned at the Zhongguancun, Beijing City on March 14th, 2002, preserving number is CGMCC0729), with TS16 is to produce bacterial strain, and its production technology is: sand pipe bacterial classification → slant activation → shake bottle investigation → seed tank culture → fermentation tank culture → filtering and concentrating → absorption → atomized drying → quality inspection → packing → finished product to put in storage.
One, medium
1, the slant activation medium adopts conventional formulation: peptone 10g/L, beef extract 5g/L, agar 2.5g/L; PH7.4～7.5,15 pound sterilization 30 minutes.
2, shake bottle and investigate medium employing conventional formulation: peptone 15g/L, yeast extract 8g/L, glucose 5g/L, NaCl5g/L; PH7.4～7.5,15 pound sterilization 30 minutes.
3, seed tank culture and fermentation tank culture all adopt the fermentation medium that enriches carbon source and nitrogenous source that contains that the present invention designs, and the contained various raw material dry matter weights of its unit volume are as follows:
Soybean cake powder 28～34g/L, corn flour 10～18g/L,
Groundnut meal 25～32g/L, fish meal 10～16g/L,
Peptone 4～8g/L, dusty yeast 1.5～4.5g/L,
FeSO
4.7H
2O?0.02～0.06g/L， ZnSO
4.7H
2O?0.04～0.1g/L，
CaCO
3?0.1～1g/L。
Soybean cake powder, corn flour, groundnut meal, fish meal require fresh not mouldy and mistake 140 mesh sieves in more than forming.
Two, fermenting and producing
1, strain inclined plane activation
Take out the sand pipe from refrigeration, the TS16 bacterial strain is inserted in the slant activation medium of above-mentioned routine, 30 ℃～32 ℃ constant temperature culture 70～75 hours.The perusal lawn is normal, and gemma forms normally.
2, shaking bottle investigates
The sand pipe bacteria suspension of activation is moved into shaking in the bottle investigation medium of above-mentioned routine, and 180～200r/min30 ℃ of shaking table cultivated 10～12 hours.The microscopic examination thalline is normal with dyeing, can enter the seeding tank enlarged culture.
3, seeding tank bacterial classification enlarged culture
Add in the seeding tank dosage bunker by the load weighted raw material of above-mentioned fermentative medium formula, add and pump into seeding tank after water stirs, dissolves, adjust pH is 8.3-8.6 before constant volume, the sterilization, press pasteurization then and sterilize, after medium is cooled off standby; Insert bacterial classification under aseptic condition, feed filtrated air, regulating ventilating ratio is 1: 0.9-1.2, operation tank pressure 0.5 kilograms per centimeter
2, cultivation temperature is 28～32 ℃, mixing speed 200～300r/min, and incubation time is 12-14 hour, can insert fermentation tank behind the microscopy and carry out fermenting and producing.
4, ferment tank is cultivated
Adopt airlift fermentor, prepare by above-mentioned fermentative medium formula and drop into behind the raw material jar, adjust pH is 8.3～8.6 before the sterilization, sterilizes then 30～40 minutes, and is standby after being cooled to 30 ℃.
Use the pressure differential method aseptic inoculation, the vigorous bacterial classification of growth in the seeding tank is inserted fermentation tank by 2～5% inoculum concentrations, regulate air capacity then, keeping ventilating ratio is 1: 1.0-1.3, tank pressure are 0.5 kilograms per centimeter
2, temperature is 29-32 ℃; Ferment after 11～14 hours, ventilating ratio is adjusted to 1: 1.5-1.7, and the fermentation power of agitator is 15kW, fermentation period is 25-28 hour.
5, post processing
When putting jar, zymotic fluid enters relay reservoir by main fermentation tank earlier, then enters ultrafiltration apparatus, and behind the circulating filtration, waste liquid drains into purification tank for liquid waste by the waste effluent flume, and the zymotic fluid after concentrating injects adsorption tanks.Then, add adsorbent, stir, pump carries out atomized drying to drying tower after 30 minutes, and dry product moisture is transported to the former powder of finished product preparation preparation unit at last and prepares packing less than 4%.
Adopt the method for utilizing novel engineering bacteria to prepare biopesticide of the present invention, its needed raw material is easy to get, and prescription is reasonable, the fermentation titer height.The present invention utilizes the biopesticide product of novel engineering bacteria preparation, except that to sensitive insects such as cabbage caterpillar, diamond-back moth, rice leaf roller, tea caterpillar, tea geometrid effectively, non-sensitive insects such as beet armyworm, prodenia litura are also had control efficiency.Constant product quality, dependable performance, result of use are good, and to the person poultry harmless, be easy to storage, transportation, easy to use.Compare with the existing product in market, have the height of tiring, relative low characteristics of use cost, the product of fermenting and producing test is tired and is reached 37678IU/mg, has increased a kind of new biopesticide for carrying out the biological control of insect pests of controlling worm with bacterium.Field demonstration test is the result show, when product was 100 μ g/ml in concentration, insecticidal effect reached more than 90%.
Disclose the method for utilizing novel engineering bacteria to prepare biopesticide of the present invention for more abundant, be illustrated below in conjunction with embodiment.
One, the prescription of medium:
1, slant activation medium: peptone 10g/L, beef extract 5g/L, agar 2.5g/L; PH7.4～7.5,15 pound sterilization 30 minutes.
2, shake bottle and investigate medium: peptone 15g/L, yeast extract 8g/L, glucose 5g/L, NaCl5g/L; PH7.4～7.5,15 pound sterilization 30 minutes.
3, fermentation medium: the contained various raw material dry matter weights of unit volume are as follows: soybean cake powder 32g/L, corn flour 14g/L, groundnut meal 32g/L, fish meal 14g/L, peptone 4g/L, dusty yeast 4.5g/L, FeSO
4.7H
2O0.04g/L, ZnSO
4.7H
2O0.08g/L, CaCO
31g/L.Soybean cake powder, corn flour, groundnut meal, fish meal require fresh not mouldy, and cross 140 mesh sieves.
Two, operating process and technical parameter
1, strain inclined plane activation: take out the TS16 bacterial classification from refrigeration, insert in the eggplant bottle slant activation medium, 30 ℃ of constant temperature culture 72 hours.The perusal lawn is full, canescence does not have assorted bacterium, no plaque or pinprick, and microscopic examination 97% above gemma, crystal come off, and do not see trophosome, and crystal is big and dyeing is dark.
2, shaking bottle investigates: the sand pipe bacteria suspension of activation is drawn 0.5ml move in the above-mentioned shake-flask culture base (the bottled 50ml medium of 500ml triangle).200r/min30 ℃ of shaking table cultivated 10 hours.Normal, consistent, the assorted bacterium of nothing that grows of microscopic examination thalline shape, and be in logarithmic growth stage, dyeing depth unanimity, spore do not occur or do not find that protoplasm is concentrated aging, can enter the seeding tank enlarged culture.
3, seeding tank bacterial classification enlarged culture: this operation employing jar appearance is 1.5 tons a seeding tank, in dosage bunker, add by the load weighted fermented and cultured based raw material of above-mentioned prescription (inventory 60%), add and pump into seeding tank after water stirs, dissolves, constant volume, transferring the medium pH value before the sterilization is 8.5, feeds live (open) steam (5 kilograms per centimeter then
2) sterilization, be heated to 121 ℃ and kept 30 minutes.Steam off is opened outlet valve, feeds filtrated air, pressurize 0.5 kilograms per centimeter
2, logical cooling water is 45 minutes in the chuck, makes material be cooled to 30 ℃ by 121 ℃.Insert the bacterial classification that shakes after bottle activates under aseptic condition, feed filtrated air, regulating ventilating ratio is 1: 0.9-1.2, operation tank pressure 0.3～0.5 kilograms per centimeter
2, 30 ± 1 ℃ of cultivation temperature, mixing speed 250r/min, incubation time 12 hours can insert fermentation tank and carry out fermenting and producing behind the microscopy.
4, ferment tank: employing jar appearance is 20 tons a airlift fermentor, and from the medium material that dosage bunker comes, advancing the preceding adjust pH of fermentation tank is 8.5, then, utilizes saturated vapor to heat, and treats that material is preheated to 80 ℃ laggard jar by normal temperature.11.5 tons of thermal materials were all sent into fermentation tank in 30 minutes, sterilized by entering steam at the bottom of the jar then, with steam material were heated to 121 ℃ in 30 minutes.At steam flow is that 0.25 ton/hour, jar temperature are to keep sterilization in 30 minutes under 121 ℃ of conditions.After being cooled to 30 ℃, use the pressure differential method aseptic inoculation, insert 0.5 ton of the interior bacterial classification of seeding tank, regulate air capacity then, keep ventilating ratio 1: 1.0-1.3, tank pressure 0.5 kilograms per centimeter
2, temperature 29-32 ℃, ferment after 13 hours, ventilating ratio is adjusted to 1: 1.5-1.7, fermentation power of agitator 15kW, fermentation period is 25-28 hour.
5, post processing: zymotic fluid enters relay reservoir by main fermentation tank through the sieve that shakes, and then the flow with 14 ton per days enters ultrafiltration apparatus, and behind the circulating filtration, waste liquid drains into purification tank for liquid waste by the waste effluent flume, and the zymotic fluid after concentrating injects adsorption tanks; Add adsorbent (diatomite) then, stir, pump carries out atomized drying to drying tower after 30 minutes, and dry product moisture is less than 4%; At last the former powder of finished product is transported to preparation preparation unit and prepares packing.Parameter control: cycles of concentration 6-7 doubly, 0.24 ton/jar of quantity of sorbent, 140 ℃ of hot air temperatures, 80 ℃ of used heat temperature, 40 ℃ of feed temperatures.
Claims (5)
1, utilize novel engineering bacteria to prepare the method for biopesticide, it is characterized in that adopting the novel engineering bacteria TS16 of the bacillus thuringiensis,Bt (Bacillusthuringiensis that obtains through induced mutations processing wild strain, preserving number:, utilize designed fermentation medium of the present invention and technological process used in the present invention to produce biopesticide CGMCC0729) for producing bacterial strain;
2, the method for utilizing novel engineering bacteria to prepare biopesticide according to claim 1 is characterized in that, the fermentation medium of use, and the contained various raw material dry matter weights of unit volume are as follows:
Soybean cake powder 28～34g/L, corn flour 10～18g/L,
Groundnut meal 25～32g/L, fish meal 10～16g/L,
Peptone 4～8g/L, dusty yeast 1.5～4.5g/L,
FeSO
4.7H
2O0.02～0.06g/L， ZnSO
4.7H
2O0.04～0.1g/L，
CaCO
30.1～1g/L；
3, the method for utilizing novel engineering bacteria to prepare biopesticide according to claim 1 and 2, it is characterized in that in seeding tank bacterial classification enlarged culture process, the medium pH value is 8.3～8.6 before the sterilization, ventilating ratio is 1: 0.9-1.2, the operation tank pressure is 0.5 kilograms per centimeter
2, cultivation temperature is 28～32 ℃, incubation time is 12-14 hour;
4, the method for utilizing novel engineering bacteria to prepare biopesticide according to claim 1 and 2, it is characterized in that in the ferment tank process, the medium pH value is 8.3～8.6 before the sterilization, it is 1 that the inoculation back keeps ventilating ratio: 1.0-1.3, tank pressure are 0.5 kilograms per centimeter
2, temperature is 29-32 ℃; Ferment after 11～14 hours, ventilating ratio is 1: 1.5-1.7, and the fermentation power of agitator is 15kW, fermentation period is 25-28 hour;
5, the method for utilizing novel engineering bacteria to prepare biopesticide according to claim 1 and 2, it is characterized in that, the fermentation medium that uses, the contained various raw material dry matter weights of unit volume are as follows: soybean cake powder 32g/L, corn flour 14g/L, groundnut meal 32g/L, fish meal 14g/L, peptone 4g/L, dusty yeast 4.5g/L, FeSO4.7H
2O0.04g/L, ZnSO
4.7H
2O0.08g/L, CaCO
31g/L.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101889586A (en) * | 2010-06-29 | 2010-11-24 | 沈阳药科大学 | Method for preparing microbe-derived bio-pesticide |
| CN101347129B (en) * | 2008-09-01 | 2011-12-14 | 佛山市正典生物技术有限公司 | Bacillus thuringiensis suspending agent for killing mosquito |
| CN111885918A (en) * | 2017-08-11 | 2020-11-03 | 埃克塞特大学 | Biological insecticide |
-
2002
- 2002-05-22 CN CN 02120513 patent/CN1459232A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347129B (en) * | 2008-09-01 | 2011-12-14 | 佛山市正典生物技术有限公司 | Bacillus thuringiensis suspending agent for killing mosquito |
| CN101889586A (en) * | 2010-06-29 | 2010-11-24 | 沈阳药科大学 | Method for preparing microbe-derived bio-pesticide |
| CN101889586B (en) * | 2010-06-29 | 2014-01-01 | 沈阳药科大学 | Method for preparing microbe-derived bio-pesticide |
| CN111885918A (en) * | 2017-08-11 | 2020-11-03 | 埃克塞特大学 | Biological insecticide |
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