CN101260144B - Inverse floatation separation purification production technique for bacillus thuringiensis berliner parasporal crystal - Google Patents
Inverse floatation separation purification production technique for bacillus thuringiensis berliner parasporal crystal Download PDFInfo
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Abstract
The invention provides a reverse flotation separation purification manufacturing process for bacillus thuringiensis parasporal crystals. The particular manufacturing process is as follows: according to weight percent, 0.2 to 0.4 percent of beef extract, 0.6 to 1.5 percent of peptone, 0.3 to 0.6 percent of sodium chloride, 1.3 to 2.5 percent of agar and 95 to 98 percent of water are weighted, and the pH value of the mixed solution is adjusted to between 7.4 and 7.6 by NaOH, and the mixed solution is sterilized at a temperature of between 100 and 130 Celsius degrees for 20 to 40 minutes, put in a culture dish and naturally cooled to room temperature, and the liquids are solidified to form the agar solid culture medium; the surface of the culture medium is inoculated according to the 3-shape, and the culture medium is put in a biochemical incubator and cultured at a temperature of between 26 and 36 DEG C for 36 to 48 hours, and thus the aseptic water lawn suspending liquid is obtained, and the aseptic water lawn suspending liquid is injected in a high efficiency micro bubble floatation column for floatation, and the liquid is centrifuged for 10 to 20 minutes, obtaining semi finished products of parasporal crystal; the semi-finished products of parasporal crystal are washed by normal saline and aseptic pure water and then centrifuged by the rotary speed of between 7000 and 9000 rpm, and thus water containing parasporal crystals is obtained, and the water containing parasporal crystals undergo the vacuum drying, so that the purified products of parasporal crystal are obtained; the purified products of parasporal crystal need to be stored at a temperature of between 20 DEG C below zero and 80 DEG C below zero. The method of the invention has the advantages of simple process, easy operation, high product purity (more than 99.99 percent) and low manufacturing cost, and is applied to the industrialized production.
Description
Technical field
The invention belongs to biological pesticide technical field, a kind of bacillus thuringiensis parasporal crystal reverse flotation separation and purification production technique is provided especially, be applicable to suitability for industrialized production.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of gram-positive genus bacillus.The thalline of Bt is a rod-short, gives birth to flagellum, single giving birth to or the formation short chain.After the nourishing body of thalline grew into certain stage, at the end formation gemma of thalline, the other end formed a kind of nearly rhombohedral protein crystal that is called as parasporal crystal, can discharge gemma and parasporal crystal after thalline breaks.
At genetically engineered, toxicologic study, the insecticidal mechanism of Bt, aspect the mankind's scientific researches such as security, all the pure article of Bt parasporal crystal have been proposed great demand.Therefore; The separation purification method of Bt parasporal crystals such as extraction process (Chinese patent 99122189.3), gradient density centrifugal method, liquid two-phase separation method, isoelectric point precipitation, alkaline lysis, supercentrifugal process and biophysics method progressively is developed (Zuo Yahui; Bacillus thuringiensis culture condition and the pre-test of crystallin method of purification; Plant protection, 1999,25 (4): 32-34; Guo Aiying, the proteic preparation method of bacillus thuringiensis crystal, food and fermentation industries, 2005,31 (8): 8-10; Zhu's bodyguard room, double-aqueous phase system separates the research of bacillus thuringiensis parasporal crystal, world's agricultural chemicals, 2000,22 (5): 39-42).But extraction process has used highly toxic organic solvent CCl
4, be unfavorable for environment protection; It is few that the gradient density centrifugal method is extracted protein content, and ultra centrifugation time is very long, about 14h, and cost is high, complicated operation; Liquid two-phase law system is complicated, and influence factor is many, is difficult to realize industriallization; Isoelectric point method has destroyed the form of parasporal crystal easily; Alkaline lysis is easy to occur the phenomenon that parasporal crystal is degraded by the basic protein lytic enzyme that self produces; The product purity that supercentrifugal process extracts is lower; The biophysics method needs special reagent, and condition is harsh, and cost is too high.Therefore, the Bt parasporal crystal still is in the personal stage of laboratory self-control, does not still have commodity and occurs.
Summary of the invention
The present invention provides a kind of Bt parasporal crystal reverse flotation separation and purification production technique; Adopt to increase a kind of Bt parasporal crystal reverse flotation separation purification method, the characteristics of this method are that technology is simple, easy handling, product purity more than 99.99%, production cost is low, be suitable for suitability for industrialized production.
The present invention adopts flotation separation method that the Bt parasporal crystal is separated with impurity such as spore, nourishing body fragments, obtains the pure article of Bt parasporal crystal, production technique (flow process is referring to accompanying drawing) as follows:
Take by weighing Carnis Bovis seu Bubali cream 0.2～0.4% by weight percentage, peptone 0.6～1.5%, sodium-chlor 0.3～0.6%; Agar 1.3～2.5%, water 95～98% is transferred pH=7.4～7.6 with NaOH; Sterilized 20～40 minutes for 100～130 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of the bacterium of going out, naturally cools to room temperature, and liquid solidifies and forms the agar solid medium; Pick the Bt bacterial classification with transfering loop, at agar solid medium surface seeding, afterwards in biochemical incubator 26～36 ℃ cultivated 36～48 hours, at this moment, on agar solid culture primary surface, form one deck lawn; With sterile purified water lawn is flushed in the container, obtains sterilized water lawn suspension liquid; Handle the lawn suspension liquid after 10～30 minutes with ultrasonic cell disruptor, this lawn suspension liquid is injected the microbubble floatation column flotation; In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with rotating speed centrifugate slurry 10～20min of 7000～9000 rev/mins, abandoning supernatant obtains the parasporal crystal work in-process afterwards; Washing parasporal crystal work in-process, centrifugal with 7000～9000 rev/mins rotating speed, obtain aqueous parasporal crystal, at-20～-80 ℃, drying is 3～4 hours under 0.1～1.0Pa vacuum degree condition, obtains the pure article of parasporal crystal.
Said at agar solid medium surface seeding, be to inoculate by " it " font.
Described flotation is that the lawn suspension liquid is progressively injected 30～100L microbubble floatation column, and the air input of flotation is 2～8L/min, flotation 10～30 minutes.
Described washing parasporal crystal work in-process are to wash the parasporal crystal work in-process 3～5 times with 0.5～1.5M NaCl saline water, and each 10～15L washs the parasporal crystal work in-process 3～5 times with sterile purified water again.
The pure article of the parasporal crystal that obtains are preserved in-20～-80 ℃ of environment.
Characteristics of the present invention are:
(1) core procedure of this technology is that reverse flotation separates parasporal crystal and impurity.Parasporal crystal is a kind of protein, and molecular weight is about 65～130KD, and the solubleness in water is very little, exists with granular form.Parasporal crystal is made up of amino acid, and its surface exists a large amount of carboxyls and amino, and therefore, the wettability of parasporal crystal is good, in the reverse flotation process, stays in the slurries;
(2) surface wettability of gemma and nourishing body fragment is poor, in the reverse flotation process, forms three-phase system with bubble, water, rises to foam layer, can separate with system well;
(3) in the process of ultrasonication fermented product suspension liquid, nourishing body is crashed to pieces, and parasporal crystal is from wherein spinning off, and simultaneously, the nutritive medium in the nourishing body gets in the slurries.These nutritive mediums contain the protein and the glucide of small molecular weight, are the whipping agents in the floatation process.Therefore, need not add whipping agent in the floatation process, so both reduce cost, avoid the introducing of impurity again;
(4) this technology uses the agar solid medium to cultivate the Bt lawn.The Bt bacterial classification is in the surface growth of agar solid medium; Form lawn after cultivating completion, with the surface of sterile purified water flushing substratum, lawn then breaks away from substratum; Form sterilized water lawn suspension liquid; The composition of agar solid medium then can not get into suspension liquid, has reduced the introducing of impurity, and the burden that has alleviated subsequent step is (if separate parasporal crystal from the Bt liquid fermentate; Because staples such as the Carnis Bovis seu Bubali cream in the liquid nutrient medium, peptone mix with Bt spore, parasporal crystal inevitably, it is very complicated that follow-up parasporal crystal purification procedures will become);
(5) in this technology owing to used saline water washing parasporal crystal, can eliminate that the interior raw albumen enzyme that produces in the fermenting process is adsorbed on the parasporal crystal surface and degraded that parasporal crystal is produced.
The present invention cultivates the Bt bacterial classification on the agar solid medium, with UW parasporal crystal and parent are broken away from, and adopts reverse flotation method that parasporal crystal is separated with impurity such as spore, nourishing body fragments, after washing, lyophilize, obtains the pure article of Bt parasporal crystal.The invention has the advantages that: technology is simple, easy handling, product purity more than 99.99%, production cost is low, be suitable for suitability for industrialized production.
Description of drawings
Fig. 1 is bacillus thuringiensis parasporal crystal reverse flotation separation and purification technological process of production figure
Embodiment
Embodiment 1:
Take by weighing Carnis Bovis seu Bubali cream 2kg, peptone 6kg, sodium-chlor 3kg; Agar 13kg, water 950kg transfers pH=7.4 with NaOH; Sterilized 20 minutes for 100 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 26 ℃ cultivated 36 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 1000L, obtain sterilized water lawn suspension liquid with the 500L sterile purified water.
Handle the lawn suspension liquid after 10 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 30L high efficiency microbubble columned pneumatic flotation machine, air input is 2L/min, flotation 10 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 7000 rev/mins rotating speed centrifugate slurry 10min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 3 times with 0.5M NaCl saline water, each 10L uses sterile purified water parasporal crystal work in-process 3 times again; Rotating speed with 7000 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-20 ℃, dry 3 hours of the vacuum tightness of 0.1Pa; Obtain the pure article 1.0kg of parasporal crystal, in-20 ℃ of environment, preserve.
Embodiment 2:
Take by weighing Carnis Bovis seu Bubali cream 4kg, peptone 15kg, sodium-chlor 6kg; Agar 25kg, water 980kg transfers pH=7.6 with NaOH; Sterilized 40 minutes for 130 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 36 ℃ cultivated 48 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 2000L, obtain sterilized water lawn suspension liquid with the 600L sterile purified water.
Handle the lawn suspension liquid after 30 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 100L high efficiency microbubble columned pneumatic flotation machine, air input is 8L/min, flotation 30 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 9000 rev/mins rotating speed centrifugate slurry 20min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 5 times with 1.5M NaCl saline water, each 15L uses sterile purified water parasporal crystal work in-process 5 times again; Rotating speed with 9000 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-80 ℃, dry 4 hours of the vacuum tightness of 1.0Pa; Obtain the pure article 1.2kg of parasporal crystal, in-80 ℃ of environment, preserve.
Embodiment 3:
Take by weighing Carnis Bovis seu Bubali cream 3kg, peptone 10kg, sodium-chlor 4.5kg; Agar 19kg, water 970kg transfers pH=7.5 with NaOH; Sterilized 30 minutes for 120 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 31 ℃ cultivated 42 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 1500L, obtain sterilized water lawn suspension liquid with the 550L sterile purified water.
Handle the lawn suspension liquid after 20 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 70L high efficiency microbubble columned pneumatic flotation machine, air input is 5L/min, flotation 20 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 8000 rev/mins rotating speed centrifugate slurry 15min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 4 times with 1.0M NaCl saline water, each 13L uses sterile purified water parasporal crystal work in-process 4 times again; Rotating speed with 8000 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-50 ℃, dry 3.5 hours of the vacuum tightness of 0.5Pa; Obtain the pure article 1.1kg of parasporal crystal, in-50 ℃ of environment, preserve.
Embodiment 4:
Take by weighing Carnis Bovis seu Bubali cream 2kg, peptone 7kg, sodium-chlor 4kg; Agar 15kg, water 970kg transfers pH=7.5 with NaOH; Sterilized 25 minutes for 120 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 28 ℃ cultivated 38 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 1000L, obtain sterilized water lawn suspension liquid with the 500L sterile purified water.
Handle the lawn suspension liquid after 30 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 100L high efficiency microbubble columned pneumatic flotation machine, air input is 8L/min, flotation 30 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 9000 rev/mins rotating speed centrifugate slurry 20min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 4 times with 1.0M NaCl saline water, each 13L uses sterile purified water parasporal crystal work in-process 4 times again; Rotating speed with 8000 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-50 ℃, dry 3.5 hours of the vacuum tightness of 0.5Pa; Obtain the pure article 1.1kg of parasporal crystal, in-50 ℃ of environment, preserve.
Embodiment 5:
Take by weighing Carnis Bovis seu Bubali cream 4kg, peptone 6kg, sodium-chlor 3kg; Agar 25kg, water 980kg transfers pH=7.6 with NaOH; Sterilized 40 minutes for 130 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 36 ℃ cultivated 48 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 2000L, obtain sterilized water lawn suspension liquid with the 600L sterile purified water.
Handle the lawn suspension liquid after 20 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 70L high efficiency microbubble columned pneumatic flotation machine, air input is 5L/min, flotation 20 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 8000 rev/mins rotating speed centrifugate slurry 15min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 3 times with 0.5M NaCl saline water, each 10L uses sterile purified water parasporal crystal work in-process 3 times again; Rotating speed with 7000 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-20 ℃, dry 3 hours of the vacuum tightness of 0.1Pa; Obtain the pure article 1.2kg of parasporal crystal, in-20 ℃ of environment, preserve.
Embodiment 6:
Take by weighing Carnis Bovis seu Bubali cream 3.1kg, peptone 12kg, sodium-chlor 5.5kg; Agar 21kg, water 970kg transfers pH=7.5 with NaOH; Sterilized 30 minutes for 120 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 31 ℃ cultivated 42 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 1500L, obtain sterilized water lawn suspension liquid with the 550L sterile purified water.
Handle the lawn suspension liquid after 10 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 30L high efficiency microbubble columned pneumatic flotation machine, air input is 2L/min, flotation 10 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 7000 rev/mins rotating speed centrifugate slurry 10min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 5 times with 1.5M NaCl saline water, each 15L uses sterile purified water parasporal crystal work in-process 5 times again; Rotating speed with 9000 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-80 ℃, dry 4 hours of the vacuum tightness of 1.0Pa; Obtain the pure article 1.2kg of parasporal crystal, in-80 ℃ of environment, preserve.
Embodiment 7:
Take by weighing Carnis Bovis seu Bubali cream 2.5kg, peptone 6kg, sodium-chlor 5.5kg; Agar 14kg, water 960kg transfers pH=7.5 with NaOH; Sterilized 22 minutes for 110 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 31 ℃ cultivated 40 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 1200L, obtain sterilized water lawn suspension liquid with the 5g0L sterile purified water.
Handle the lawn suspension liquid after 15 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 50L high efficiency microbubble columned pneumatic flotation machine, air input is 4L/min, flotation 18 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 7800 rev/mins rotating speed centrifugate slurry 13min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 4 times with 0.8M NaCl saline water, each 11L uses sterile purified water parasporal crystal work in-process 5 times again; Rotating speed with 8200 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-40 ℃, dry 3.3 hours of the vacuum tightness of 0.3Pa; Obtain the pure article 1.1kg of parasporal crystal, in-60 ℃ of environment, preserve.
Embodiment 8:
Take by weighing Carnis Bovis seu Bubali cream 3.6kg, peptone 9kg, sodium-chlor 6kg; Agar 20kg, water 980kg transfers pH=7.6 with NaOH; Sterilized 30 minutes for 130 ℃; Liquid after in clean bench, will sterilizing is poured in the big petridish of 20 bacterium of having gone out (diameter 0.5m), naturally cools to room temperature, and liquid solidifies and forms the agar solid medium.Pick a small amount of Bt bacterial classification with transfering loop, " it " font inoculation on agar solid culture primary surface, afterwards in biochemical incubator 29 ℃ cultivated 30 hours.At this moment, on agar solid culture primary surface, form one deck lawn, lawn is flushed in the container of 1600L, obtain sterilized water lawn suspension liquid with the 520L sterile purified water.
Handle the lawn suspension liquid after 28 minutes with ultrasonic cell disruptor, this lawn suspension liquid is progressively injected the flotation of 90L high efficiency microbubble columned pneumatic flotation machine, air input is 8L/min, flotation 10 minutes.In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with 7200 rev/mins rotating speed centrifugate slurry 18min, abandoning supernatant obtains the parasporal crystal work in-process afterwards.
Wash the parasporal crystal work in-process 5 times with 1.2M NaCl saline water, each 14L uses sterile purified water parasporal crystal work in-process 3 times again; Rotating speed with 8800 rev/mins is centrifugal; Obtain aqueous parasporal crystal ,-80 ℃, dry 3.5 hours of the vacuum tightness of 0.8Pa; Obtain the pure article 1.1kg of parasporal crystal, in-50 ℃ of environment, preserve.
Claims (5)
1. a bacillus thuringiensis parasporal crystal reverse flotation separation and purification production technique is characterized in that: take by weighing Carnis Bovis seu Bubali cream 0.2～0.4% by weight percentage, peptone 0.6～1.5%; Sodium-chlor 0.3～0.6%, agar 1.3～2.5%, surplus is a water; Transfer pH=7.4～7.6,100～130 ℃ sterilization 20～40 minutes with NaOH, the liquid after in clean bench, will sterilizing is poured in the big petridish of the bacterium of going out; Naturally cool to room temperature, liquid solidifies and forms the agar solid medium; Pick the Bt bacterial classification with transfering loop, at agar solid medium surface seeding, afterwards in biochemical incubator 26～36 ℃ cultivated 36～48 hours; With sterile purified water lawn is flushed in the container, obtains sterilized water lawn suspension liquid; Handle the lawn suspension liquid after 10～30 minutes with ultrasonic cell disruptor, this lawn suspension liquid is injected the microbubble floatation column flotation; In this reverse flotation process, gemma and nourishing body fragment get into foam layer and the disengaging system, and parasporal crystal is then stayed in the liquid slurry, and with rotating speed centrifugate slurry 10～20min of 7000～9000 rev/mins, abandoning supernatant obtains the parasporal crystal work in-process afterwards; Washing parasporal crystal work in-process, centrifugal with 7000～9000 rev/mins rotating speed, obtain aqueous parasporal crystal, at-20～-80 ℃, drying is 3～4 hours under 0.1～1.0Pa vacuum degree condition, obtains the pure article of parasporal crystal.
2. production technique as claimed in claim 1 is characterized in that, on agar solid culture primary surface, inoculates by " it " font.
3. production technique as claimed in claim 1 is characterized in that, described flotation is that the lawn suspension liquid is progressively injected 30～100L microbubble floatation column, and the air input of flotation is 2～8L/min, flotation 10～30 minutes.
4. production technique as claimed in claim 1; It is characterized in that; Described washing parasporal crystal work in-process are to wash the parasporal crystal work in-process 3～5 times with 0.5～1.5M NaCl saline water, and each 10～15L washs the parasporal crystal work in-process 3～5 times with sterile purified water again.
5. production technique as claimed in claim 1 is characterized in that, the pure article of the parasporal crystal that obtains are preserved in-20～-80 ℃ of environment.
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| CN102598909A (en) * | 2012-03-02 | 2012-07-25 | 天津师范大学 | Method for regulating activity of soil matrix enzyme with Bacillus thuringiensis fermentation liquid |
| CN112175051B (en) * | 2020-09-03 | 2022-05-20 | 武汉科诺生物科技股份有限公司 | Rapid purification method of low-content Bt crystal protein |
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| CN1348490A (en) * | 1999-02-26 | 2002-05-08 | 美国辉瑞有限公司 | Method for the purification, recovery, and sporulation of cysts and oocysts |
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