CN102027999A - Bacillus thuringiensis wettable powder containing pullulan polysaccharide and preparation method thereof - Google Patents
Bacillus thuringiensis wettable powder containing pullulan polysaccharide and preparation method thereof Download PDFInfo
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- CN102027999A CN102027999A CN2010105490390A CN201010549039A CN102027999A CN 102027999 A CN102027999 A CN 102027999A CN 2010105490390 A CN2010105490390 A CN 2010105490390A CN 201010549039 A CN201010549039 A CN 201010549039A CN 102027999 A CN102027999 A CN 102027999A
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Abstract
The invention provides bacillus thuringiensis wettable powder containing pullulan polysaccharide and a preparation method thereof, relating to powder and a preparation method thereof. The invention solves the problems that the quality guarantee period and the valid period of the powder active ingredients prepared by the traditional technology are short, and the preparation process is single and dated. The bacillus thuringiensis wettable powder containing pullulan polysaccharide of the invention comprises a spore and parasporal crystal mixture, a carrier, a wetting agent and a dispersing agent. The preparation method comprises the steps of: (1) preparing seed bacteria liquid; (2) fermenting and cultivating the seed bacteria liquid to obtain fermentation liquid; (3) concentrating the fermentation liquid to obtain spore and parasporal crystal; (4) adding the carrier and NaCl to the spore and parasporal crystal mixture and blending uniformly; and (5) adding the wetting agent and the dispersing agent, blending uniformly, drying and grinding to obtain the bacillus thuringiensis wettable powder containing pullulan polysaccharide. The bacillus thuringiensis in the powder obtained in the invention has high activity, long quality guarantee period and valid period, and can be widely applied to the field of pest control in the field.
Description
Technical field
The present invention relates to a kind of pulvis and preparation method thereof.
Background technology
One of main reason that causes crop failure now is an agricultural insect pest, though traditional chemical pesticide has certain preventive and therapeutic effect to insect, but the negative effect that it brings also is on the rise, reduce drug effect thereby insect is developed immunity to drugs, increase the control cost, toxicity is bigger, not only harmful to people and animals, and pollute the environment, killed natural enemies, even destroy the ecological balance.Microorganism insecticide has then overcome above shortcoming, and have that selectivity is strong, advantage such as safety, raw material are simple, therefore become one of research focus of crop pests control.(Bacillus thuringiensis is that the maximum of research also are most popular biological prevention and control agents Bt) to bacillus thuringiensis in the microorganism insecticide of studying at present.
Thuringiensis cladosporioides bacillus insecticide is since France in 1938 comes out, obtained significant progress, the formulation of exploitation has also had a variety of, comprise pulvis, suspending agent, wetting powder, finish, oil emu, granule, tablet, sustained release agent, biological coating agent or the like, nearly 100 years research and development make ripe gradually perfect with the microorganism insecticide of bacillus thuringiensis microbial source.Field control effect reaches and in China kind more than 20 is just arranged more than 80%.But in application process, also there is main onset composition instability, causes great number of issues such as shelf-life of active component and lasting period difference.
Contained parasporal crystal of Su Yun bronze agent and gemma exist with dry powder, have characteristics such as storage tolerance, convenient transportation, usability are strong, the height of tiring.But because pulvis is difficult moistening by water, also be difficult for disperseing and being suspended in the water, the dilution spraying is difficult.Thuringiensis pulvis water content requires below 4%, and the composition of could remaining valid the long period like this is in stable state.
In addition, single, the outmoded problem of preparation method's ubiquity of bacillus thuringiensis pulvis, production technology is also backward relatively, causes uncertain, the main onset composition of physiological detection index of product also unstable, and this makes that the development of microbial bactericide is hindered.
At present pulvis just towards height tire, the direction of multi-functional (wide spectrum, synergy, delaying drug resistance, reduce cost) develops, the improved work of formulation is primarily aimed at these deficiencies and carries out.The pulullan polysaccharide tool is white non-hygroscopic powder, nontoxic, no teratogenesis, tasteless, be soluble in cold water, have unique character such as water-soluble, film forming, one-tenth fibroid, water imbibition, therefore utilize pulullan polysaccharide more and more to be subject to people's attention as the research of drug delivery system carrier material; And in production technology, then need to select novel form, new auxiliary and new compound, and to improve the dispersiveness and the tack of active ingredient, prolong the shelf-life of active component, also be a great problem that needs to be resolved hurrily at present.
Summary of the invention
The present invention is in order to solve the existing main active ingredient instability of bacillus thuringiensis pulvis, the storage life and the lasting period of active component are poor, the problem that preparation technology is single, outmoded, and a kind of bacillus thuringiensis wetting powder that contains pulullan polysaccharide and preparation method thereof is provided.
The bacillus thuringiensis wetting powder that the present invention is a kind of to contain pulullan polysaccharide is made up of 50%～70% gemma and parasporal crystal mixture, 0.5%～5% carrier, 5%～20% wetting agent, 5%～20% dispersant and 1%～5% NaCl by weight percentage; Wherein the quantity ratio of gemma and parasporal crystal is 0.8～1.2: 1 in gemma and the parasporal crystal mixture, and dispersant is a pulullan polysaccharide.
A kind of preparation method who contains the bacillus thuringiensis wetting powder of pulullan polysaccharide of the present invention realizes according to the following steps: the bacillus thuringiensis of hiding of, going bail for is inoculated on the LB solid slant medium, under 30 ℃ of conditions, cultivate 24h, and then transferring in 5mL LB liquid nutrient medium with oese picking lawn on the LB solid slant medium, under 30 ℃ of conditions, rotating speed with 200r/min shakes bacterium 12h, as bacillus thuringiensis kind daughter bacteria liquid; Two, will plant daughter bacteria liquid by volume mark be 2% to be inoculated in the 50mL fermentation medium, under 30 ℃ of conditions, shake bacterium 40～50h with the rotating speed of 200r/min, be 45%～55% until the bacillus exfoliation amount, obtain the bacillus thuringiensis zymotic fluid; Three, with 1mol/L HCl solution zymotic fluid is transferred to pH value 4.5, divide then to install in 2 centrifuge tubes,, reclaim precipitation then with the centrifugal 20min of 5000～8000r/min rotating speed, obtain gemma and parasporal crystal mixture, the quantity ratio of gemma and parasporal crystal is 0.8～1.2: 1; Four, add 50%～70% gemma and parasporal crystal mixture, 0.5%～5% carrier and 1%～5% NaCl by weight percentage, stir; Five, add 5%～20% wetting agent and 5%～20% dispersant behind the mixing by weight percentage, stir, oven dry is to moisture mass percent 3%～taking-up in 4% o'clock under 50～70 ℃ of conditions, grind then, through 75 μ m sieve, obtain a kind of bacillus thuringiensis wetting powder that contains pulullan polysaccharide again; Wherein, dispersant is a pulullan polysaccharide in the step 5.
Because the good film forming characteristics of pulullan polysaccharide more helps to keep the activity of bacillus thuringiensis.
Bacillus thuringiensis living spores content 8.0～9.2 * 10 in the bacillus thuringiensis wetting powder that contains pulullan polysaccharide of the present invention's preparation
9Individual/g, pH7.2; By 75 μ m sieve back fineness is 99.9%, and the national standard fineness is 98% at present; Suspensibility (being effective content) 72% is higher than the suspensibility 70% of present national standard; Wetting time is 123s, obviously shortens in national standard wetting time 180s; Moisture percetage by weight 3.8%, national standard are 4%; The bacillus thuringiensis wetting powder of the pulullan polysaccharide that storage life obtained with the present invention in the time of 6 months is handled, corrected mortality is 96%, the active height of bacillus thuringiensis only is 90% and use the bacillus thuringiensis pulvis corrected mortality of other dispersants preparations.
The present invention is incorporated in the preparation process of bacillus thuringiensis wetting powder as carrier, neopelex or lauryl sodium sulfate as wetting agent, pulullan polysaccharide artificial zeolite, kaolin or diatomite as dispersant, the bacillus thuringiensis wetting powder that contains pulullan polysaccharide of preparation has good storage life and lasting period, add novel form, new auxiliary and new compound, remedied single, the outmoded defective of prior art for preparing technology.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises any combination between each embodiment.
Embodiment one: the bacillus thuringiensis wetting powder that present embodiment is a kind of to contain pulullan polysaccharide is made up of 50%～70% gemma and parasporal crystal mixture, 0.5%～5% carrier, 5%～20% wetting agent, 5%～20% dispersant and 1%～5% NaCl by weight percentage; Wherein the quantity ratio of gemma and parasporal crystal is 0.8～1.2: 1 in gemma and the parasporal crystal mixture, and dispersant is a pulullan polysaccharide.
Embodiment two: what present embodiment and embodiment one were different is that described carrier is artificial zeolite, kaolin or diatomite.Other is identical with embodiment one.
Embodiment three: what present embodiment was different with embodiment one or two is that described wetting agent is neopelex or lauryl sodium sulfate.Other is identical with embodiment one or two.
Embodiment four: present embodiment is a kind of preparation method who contains the bacillus thuringiensis wetting powder of pulullan polysaccharide realize according to the following steps: the bacillus thuringiensis of hiding of, going bail for is inoculated on the LB solid slant medium, under 30 ℃ of conditions, cultivate 24h, and then transferring in 5mL LB liquid nutrient medium with oese picking lawn on the LB solid slant medium, under 30 ℃ of conditions, rotating speed with 200r/min shakes bacterium 12h, as bacillus thuringiensis kind daughter bacteria liquid; Two, will plant daughter bacteria liquid by volume mark be 2% to be inoculated in the 50mL fermentation medium, under 30 ℃ of conditions, shake bacterium 40～50h with the rotating speed of 200r/min, be 45%～55% until the bacillus exfoliation amount, obtain the bacillus thuringiensis zymotic fluid; Three, with 1mol/L HCl solution zymotic fluid is transferred to pH value 4.5, divide then to install in 2 centrifuge tubes,, reclaim precipitation then with the centrifugal 20min of 5000～8000r/min rotating speed, obtain gemma and parasporal crystal mixture, the quantity ratio of gemma and parasporal crystal is 0.8～1.2: 1; Four, add 50%～70% gemma and parasporal crystal mixture, 0.5%～5% carrier and 1%～5% NaCl by weight percentage, stir; Five, add 5%～20% wetting agent and 5%～20% dispersant behind the mixing by weight percentage, stir, oven dry is to moisture mass percent 3%～taking-up in 4% o'clock under 50～70 ℃ of conditions, grind then, through 75 μ m sieve, obtain a kind of bacillus thuringiensis wetting powder that contains pulullan polysaccharide again; Wherein, dispersant is a pulullan polysaccharide in the step 5.
Bacillus thuringiensis is bought from Institute of Microorganism, Academia Sinica in the present embodiment.
Embodiment five: what present embodiment and embodiment four were different is: the carrier in the step 4 is artificial zeolite, kaolin or diatomite.Other step is identical with embodiment four with parameter.
Embodiment six: what present embodiment was different with embodiment four or five is: the wetting agent in the step 5 is neopelex or lauryl sodium sulfate.Other step is identical with embodiment four or five with parameter.
Embodiment seven: what present embodiment and embodiment six were different is that the bacillus thuringiensis fermentation medium is with glucose, soybean cake powder, dusty yeast, fish meal, peptone, K in the step 2
2HPO
4And CaCl
2The preparation medium that is dissolved in the water, glucose 20g/L wherein, soybean cake powder 20g/L, dusty yeast 16g/L, fish meal 20g/L, peptone 10g/L, K
2HPO
420g/L, CaCl
230g/L, adjust pH to 6.8～7.0, in the 250mL triangular flask of packing into, 30min sterilizes under 0.14Mpa pressure.Other step is identical with embodiment six with parameter.
Embodiment eight: present embodiment is a kind of preparation method who contains the bacillus thuringiensis wetting powder of pulullan polysaccharide realize according to the following steps: the bacillus thuringiensis of hiding of, going bail for is inoculated on the LB solid slant medium, under 30 ℃ of conditions, cultivate 24h, and then transferring in 5mL LB liquid nutrient medium with oese picking lawn on the LB solid slant medium, under 30 ℃ of conditions, rotating speed with 200r/min shakes bacterium 12h, as bacillus thuringiensis kind daughter bacteria liquid; Two, will plant daughter bacteria liquid by volume mark be 2% to be inoculated in the fermentation medium of 50mL, under 30 ℃ of conditions, shake bacterium 48h with the rotating speed of 200r/min, be 50% until the bacillus exfoliation amount, obtain the bacillus thuringiensis zymotic fluid; Three, with 1mol/L HCl solution zymotic fluid is transferred to pH value 4.5, divide then to install in 2 centrifuge tubes,, reclaim precipitation then with the centrifugal 20min of 6000r/min rotating speed, obtain gemma and parasporal crystal mixture, the quantity ratio of gemma and parasporal crystal is 0.8～1.2: 1; Four, add 50%～70% gemma and parasporal crystal mixture, 0.5%～5% carrier and 1%～5% NaCl by weight percentage, stir; Five, add 5%～20% wetting agent and 5%～20% dispersant behind the mixing by weight percentage, stir, oven dry 5h took out at 4% o'clock to water content under 55 ℃ of conditions, grind dried mixture with mortar then, through 75 μ m sieve, obtain a kind of bacillus thuringiensis wetting powder that contains pulullan polysaccharide again; Wherein, the carrier in the step 4 is artificial zeolite, kaolin or diatomite; Wetting agent in the step 5 is neopelex or lauryl sodium sulfate, and dispersant is a pulullan polysaccharide.
Carrier artificial zeolite, kaolin or diatomite in the present embodiment grinds repeatedly with mortar in use, through 75 μ m sieve, obtains fine powder then.
Embodiment nine: the present embodiment utilization is with zymotic fluid and three kinds of different carriers artificial zeolites of a collection of bacillus thuringiensis; kaolin or diatomite are mixed with the bacillus thuringiensis wetting powder that becomes to contain pulullan polysaccharide respectively; as shown in table 1; dry back statistics living spores content; draw carrier after the mensuration gemma in the bacillus thuringiensis wetting powder is had protective effect; and different carriers is variant to the protective effect of bacillus thuringiensis; the protective effect to bacillus thuringiensis when percentage by weight 1.0%～3.0% concentration reaches peak as kaolin and diatomite, and artificial zeolite then just can reach higher protective effect under the concentration of percentage by weight 5.0%.
In addition, the protection of bacillus thuringiensis wetting powder also is not quite similar under variable concentrations with a kind of carrier, table 1 is the influence to gemma of different carriers kind and content.
Table 1
By shown in the table 1, kaolin is extremely strong in percentage by weight 1.0%～3.0% o'clock protective effect, but when percentage by weight reached 5.0%, it was to the almost unprotect effect of bacillus thuringiensis wetting powder.
Embodiment ten: present embodiment has been investigated and has not been added people's wetting agent and add the influence of wetting agent to the Su Yun gold moistening time of wetting powder of different carriers preparation, as contrast 1 in the table 2 is water and carrier biased sample, contrast 2 is sterilization blank fermentation medium in back and carrier biased sample, and contrast 3 is zymotic fluid and carrier biased sample.
Table 2
As shown in Table 2, the sample wetting time that has added wetting agent obviously is less than contrast 1,2 and 3, and wherein the effect of wetting agent neopelex is better.
Embodiment 11: present embodiment is used the wetting powder of different dispersant preparations, and dispersant is respectively pulullan polysaccharide and sodium cellulose glycolate.With pullulan-containing bacillus thuringiensis wetting powder with when containing sodium cellulose glycolate bacillus thuringiensis wetting powder and preserving 1 day, 3 months and 6 months respectively, utilize diamond-back moth 2 instar larvaes to carry out biological activity determination.Concrete assay method is: the bacillus thuringiensis wetting powder that contains different dispersants that will prepare with leaf dipping method is diluted to 10 respectively
-3Evenly spread upon on the fresh Brussels sprouts leaf, put into the 9cm plate after drying naturally, bottom moisture releasing filter paper, every ware inserts 20 of larvas, each is handled and repeats 3 times, spreads upon on the fresh Brussels sprouts leaf with sterile water simultaneously and does blank, and handling and being placed on temperature is 26 ± 1 ℃, light application time reaches in the biochemical incubator of 14h cultivates, observe the upgrowth situation of larva behind the 72h, record borer population alive, calculation correction lethality.
Table 3
Testing result is as shown in table 3, and the result shows that the lasting period of the bacillus thuringiensis wetting powder of pullulan-containing under the equal preservation condition is higher than and contains sodium cellulose glycolate bacillus thuringiensis wetting powder.When preserving 6 months, the corrected mortality of pullulan-containing bacillus thuringiensis wetting powder is 96%, and the corrected mortality that contains sodium cellulose glycolate bacillus thuringiensis wetting powder only is 90%, simultaneously illustrate that also pulullan polysaccharide has good film forming characteristics as dispersant, helps to keep the activity of bacillus thuringiensis.
Claims (7)
1. bacillus thuringiensis wetting powder that contains pulullan polysaccharide is characterized in that the described bacillus thuringiensis wetting powder that contains pulullan polysaccharide is made up of 50%～70% gemma and parasporal crystal mixture, 0.5%～5% carrier, 5%～20% wetting agent, 5%～20% dispersant and 1%～5% NaCl by weight percentage; Wherein the quantity ratio of gemma and parasporal crystal is 0.8～1.2: 1 in gemma and the parasporal crystal mixture, and dispersant is a pulullan polysaccharide.
2. a kind of bacillus thuringiensis wetting powder that contains pulullan polysaccharide according to claim 1 is characterized in that described carrier is artificial zeolite, kaolin or diatomite.
3. a kind of bacillus thuringiensis wetting powder that contains pulullan polysaccharide according to claim 1 and 2 is characterized in that described wetting agent is neopelex or lauryl sodium sulfate.
4. prepare the described a kind of method that contains the bacillus thuringiensis wetting powder of pulullan polysaccharide of claim 1, it is characterized in that it realizes according to the following steps: the bacillus thuringiensis of hiding of, going bail for is inoculated on the LB solid slant medium, under 30 ℃ of conditions, cultivate 24h, and then on LB solid slant medium, transfer in the 5mLLB liquid nutrient medium with oese picking lawn, under 30 ℃ of conditions, rotating speed with 200r/min shakes bacterium 12h, as bacillus thuringiensis kind daughter bacteria liquid; Two, will plant daughter bacteria liquid by volume mark be 2% to be inoculated in the 50mL fermentation medium, under 30 ℃ of conditions, shake bacterium 40～50h with the rotating speed of 200r/min, be 45%～55% until the bacillus exfoliation amount, obtain the bacillus thuringiensis zymotic fluid; Three, with 1mol/LHCl solution zymotic fluid is transferred to pH value 4.5, divide then to install in 2 centrifuge tubes,, reclaim precipitation then with the centrifugal 20min of 5000～8000r/min rotating speed, obtain gemma and parasporal crystal mixture, the quantity ratio of gemma and parasporal crystal is 0.8～1.2: 1; Four, add 50%～70% gemma and parasporal crystal mixture, 0.5%～5% carrier and 1%～5% NaCl by weight percentage, stir; Five, add 5%～20% wetting agent and 5%～20% dispersant behind the mixing by weight percentage, stir, oven dry is to moisture mass percent 3%～taking-up in 4% o'clock under 50～70 ℃ of conditions, grind then, through 75 μ m sieve, obtain a kind of bacillus thuringiensis wetting powder that contains pulullan polysaccharide again; Wherein, the dispersant in the step 5 is a pulullan polysaccharide.
5. a kind of preparation method who contains the bacillus thuringiensis wetting powder of pulullan polysaccharide according to claim 4 is characterized in that the carrier in the step 4 is artificial zeolite, kaolin or diatomite.
6. according to claim 4 or 5 described a kind of preparation methods that contain the bacillus thuringiensis wetting powder of pulullan polysaccharide, it is characterized in that the wetting agent in the step 5 is neopelex or lauryl sodium sulfate.
7. a kind of preparation method who contains the bacillus thuringiensis wetting powder of pulullan polysaccharide according to claim 6 is characterized in that the bacillus thuringiensis fermentation medium is with glucose, soybean cake powder, dusty yeast, fish meal, peptone, K in the step 2
2HPO
4And CaCl
2The preparation medium that is dissolved in the water, glucose 20g/L wherein, soybean cake powder 20g/L, dusty yeast 16g/L, fish meal 20g/L, peptone 10g/L, K
2HPO
420g/L, CaCl
230g/L, adjust pH to 6.8～7.0, in the 250mL triangular flask of packing into, 30min sterilizes under 0.14Mpa pressure.
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Cited By (7)
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| CN107926984A (en) * | 2017-11-29 | 2018-04-20 | 江苏源洁节能环保有限公司 | A kind of biological insecticides for water environment |
| CN108124876A (en) * | 2018-01-09 | 2018-06-08 | 广东益林科技有限公司 | A kind of pharmaceutical composition for preventing Mikania micrantha and its preparation production method and application |
| CN110089526A (en) * | 2019-04-24 | 2019-08-06 | 中国农业科学院植物保护研究所 | Plant polyose is promoting application and composition in thuringiensis prevention and treatment insect |
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| CN112978940A (en) * | 2021-04-22 | 2021-06-18 | 广州市点滴生物科技有限公司 | Organic sewage biological fermentation treating agent and preparation method thereof |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
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| CN112978940B (en) * | 2021-04-22 | 2021-07-16 | 广州市点滴生物科技有限公司 | Organic sewage biological fermentation treating agent and preparation method thereof |
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Application publication date: 20110427 |