CN107090420A - A kind of fermentation culture method of bacillus thuringiensis - Google Patents
A kind of fermentation culture method of bacillus thuringiensis Download PDFInfo
- Publication number
- CN107090420A CN107090420A CN201710420533.9A CN201710420533A CN107090420A CN 107090420 A CN107090420 A CN 107090420A CN 201710420533 A CN201710420533 A CN 201710420533A CN 107090420 A CN107090420 A CN 107090420A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- culture medium
- bacillus thuringiensis
- medium
- cfu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of culture medium and fermentation process of the bacillus thuringiensis new strains for killing grub class pest, so that bacillus thuringiensis new strains Bacillus thuringiensis B Y7 1 have more preferable ferment effect.The fermentation medium of the present invention, it is the 31g/L of soybean cake powder 21, the 24g/L of cornstarch 16, the 0.8g/L of calcium carbonate 0.4 that it, which is formulated, dusty yeast 3 7g/L, dipotassium hydrogen phosphate 0.3g/L, magnesium sulfate 0.2g/L, zinc sulfate 0.2g/L, glucose 1g/L, sodium chloride 2g/L, manganese sulfate 0.02g/L.Cultivation results show that culture medium provided by the present invention has good ferment effect.
Description
Technical field
The invention belongs to microbial fermentation culture technique field, and in particular to a kind of golden gemma of the Su Yun for killing grub class pest
The production gemma and the liquid submerged femrentation culturing method of insecticidal crystal protein of bacillus strain.
Background technology
Bacillus thuringiensis (Bacillus thuringiensis, abbreviation Bt) is that a kind of extremely extensive leather of distribution is blue
Family name's positive bacteria, it is while gemma is formed, and can produce has specific activity to various insects, mite class and protozoan etc.
The parasporal crystal of property of protein, due to its insecticidal crystal protein (Insecticidal crystal proteins, abbreviation ICPs)
It is efficiently single-minded to target pest, it is safe and environmentally safe to non-target organism.Therefore, Bt be widely used in preventing and treating agricultural,
Insect in terms of warehousing and health, is that yield is maximum both at home and abroad at present, application most wide microbial insecticide, its desinsection
Crystalline protein is also to be most widely used and most potential class insect resistance protein at present.Before 1980s, relevant Bt
Research and application have focused largely in preventing and treating to lepidoptera pest.
Grub is the general designation of coleoptera scarab beetle Superfamily larva, is that most wide and most heavy one of causing harm is distributed in subterranean pest-insect is big
Monoid, annual about 100,000,000 mu of the occurring area in the whole nation, chemical pesticide occupies critical role, but long-term useization in terms of grub control
The problems such as environmental pollution that medicine of learning to farm is brought, disruption of ecological balance, the residual exceeded, person poultry poisoning of agriculture, pest resistance to insecticide strengthen highlights, therefore
The biological control of grub receives much concern.The immense success obtained based on bacillus thuringiensis in terms of lepidoptera pest is prevented and treated,
People begin to focus on the bacillus thuringiensis research of control of grubs, and having filtered out some has the bacterium of preferable biological and ecological methods to prevent plant disease, pests, and erosion potential to grub
Strain, such as HBF-1 (Feng Shuliang, 2000), Bt185 (Yu etc., 2006) etc..Research shows, bacillus thuringiensis B-Y7-1
Bacterial strain can produce gemma and parasporal crystal, and gemma is in oval, and parasporal crystal protein molecular weight is 133.3kDa, and isoelectric point is
4.68；The bacterial strain has special insecticidal activity to a variety of grubs, and viable bacteria and crystalline protein are the effective active composition of desinsection, are had
Important Development volue and good application prospect.The premise of biocontrol microorganisms development and application is efficient biocontrol microorganisms fermented and cultured, because
Biocontrol bacterial strain is different with culture purpose, and fermentation medium composition, condition of culture, fermented and cultured mode are also different.At present about B-
Sporulation and crystalline protein changing rule are unclear during Y7-1 strain culturings, fermentation production efficiency is not high, gemma and
Crystalline protein yield is relatively low, constrains the large-scale production and exploitation of B-Y7-1 bacterial strains.Therefore, B-Y7-1 bacterial strains are developed
Fermented and cultured technology, obtains substantial amounts of gemma or parasporal crystal protein, is the basis that B-Y7-1 bacterial strains are developed, is also preparation
Production and the premise of application, the preventing and treating for grub category subterranean pest-insect are significant.
The content of the invention
It is an object of the invention to provide a kind of culture medium and hair of the bacillus thuringiensis new strains for killing grub class pest
Fermenting process, so that bacillus thuringiensis new strains Bacillus thuringiensis B-Y7-1 have more preferable fermentation
Effect.
Present invention firstly provides a kind of fermentation medium suitable for bacillus thuringiensis B-Y7-1 bacterial strains, it is formulated such as
Under：
Soybean cake powder 21-31g/L, cornstarch 16-24g/L, calcium carbonate 0.4-0.8g/L, dusty yeast 3-7g/L, phosphoric acid
Hydrogen dipotassium 0.3g/L, magnesium sulfate 0.2g/L, zinc sulfate 0.2g/L, glucose 1g/L, sodium chloride 2g/L, manganese sulfate 0.02g/L.
Preferably, described culture medium, it is soybean cake powder 25g/L, cornstarch 24g/L, dipotassium hydrogen phosphate that it, which is formulated,
0.3g/L, magnesium sulfate 0.2g/L, zinc sulfate 0.2g/L, calcium carbonate 0.6g/L, dusty yeast 5.0g/L, glucose 1g/L, sodium chloride
2g/L, manganese sulfate 0.02g/L.
Bacillus thuringiensis new strains Bacillus thuringiensis B-Y7-1 of the present invention, in
It is preserved in China typical culture collection center on July 26th, 2011, deposit number is CCTCC No.M2011267.
Described culture medium, its pH is 7.0.
Another aspect of the present invention provides a kind of small-scale processes for cultivating bacillus thuringiensis B-Y7-1 bacterial strains, described
Method include the steps：
Step 1) culture medium preparation：
Fermentation medium is prepared, it is 6.5-8.0 to adjust initial pH；
Step 2) packing：
The culture medium of preparation is loaded into triangular flask, liquid amount is the 1/5~1/2 of triangular flask dischargeable capacity；
Step 3) sterilizing：
121 DEG C of high pressure steam sterilizations 30 minutes；
Step 4) liquid spawn preparation：
The bacillus thuringiensis single bacterium colony of picking activation, is inoculated in LB fluid nutrient mediums, 28 DEG C of shaken cultivation 18h,
Thalline quantity reaches 108Cfu/mL, is used as seed liquor；
Step 5) inoculation：
According to 1%~7% inoculum concentration, sterile working is inoculated in step 3) sterilizing after culture medium in；
Step 6) fermentation：
It is placed in complete warm shaking table, 27 DEG C, 8~72h of 150r/min shaken cultivations.
Another aspect of the present invention provides a kind of ferment tank method for cultivating bacillus thuringiensis B-Y7-1 bacterial strains：
Step 1) culture medium preparation：
DEG C fermentation medium is prepared, it is 7.2 to adjust initial pH；
Step 2) sterilizing：
Fermentation medium is added in air blow tank with machinery agitation, coefficient 75%, 121 DEG C, the real 30min that disappears；
Step 3) inoculation：
By thalline quantity 108Cfu/mL seed culture fluids, by 3% inoculum concentration, are inoculated into fermentation tank；
Step 4) fermentation：
Ventilation ratio 1:0.8~1:1.2,250r/min, 27 DEG C of fermentations, froth breaking is automatically controlled by defoamer of soya-bean oil, is fermented
During pH it is natural or control pH to be 7.0, fermentation time 8-72 hours.
It is preferred that shake flask fermentation method, fermentation medium, it is 7.0 to adjust initial pH, and liquid amount is triangular flask dischargeable capacity
1/5,121 DEG C of high pressure steam sterilizations 30 minutes.Using LB cultures 18h, thalline quantity 108Cfu/mL nutrient solutions as seed,
It is 3% that it, which is inoculated with inoculum concentration,；27 DEG C, 150r/min shaken cultivations, 40h albumin crystal content highests of fermenting, reach 3.931mg/
ML, Number of spores is more than 1 × 1010cfu/mL。
It is preferred that ferment tank method：Fermentation medium, it is 7.2 to adjust initial pH, adds mechanical agitation ventilating fermentation
In tank, coefficient 75%, disappear 30min in fact.Using LB cultures 18h, thalline quantity 108Cfu/mL nutrient solutions as seed, its
It is 3% to be inoculated with inoculum concentration；Ventilation ratio 1:1.2,250r/min, 27 DEG C of fermentations, froth breaking is automatically controlled by defoamer of soya-bean oil, is fermented
During control pH to be 7.0, fermentation time is 32 hours, and protein yield is 2.57mg/mL, and gemma amount is more than 4 × 109cfu/
mL。
Brief description of the drawings
Fig. 1：Different basal medium bacillus thuringiensis spore production analysis charts
Fig. 2：Different initial pH spore production analysis charts；
Fig. 3：Different liquid amount spore production analysis charts；
Fig. 4：Different vaccination amount spore production analysis chart；
Fig. 5：Different fermentations time Number of spores figure；
Fig. 6：Different fermentations time crystalline protein content figure；
In above-mentioned figure, wherein capitalization (A, B, C, D) represents the significance level of difference (p<0.01), behind each processing
It is alphabetical identical when for not significantly, only letter completely it is different when it is just extremely notable；Lowercase (a, b, c, d) represents that difference shows
Work level (p<0.05) for significantly, ability is not notable when only letter is complete different when alphabetical identical behind, each handling.
Embodiment
The screening of the bacillus thuringiensis bacterial strain B-Y7-1 basal mediums of embodiment 1
Applicant has found during early-stage Study, long-acting carbon source cornstarch 5g~25g/L, long-acting nitrogen source soybean cake powder
10g~35g/L, quick-acting nitrogen source dusty yeast 3g~6g/L, dipotassium hydrogen phosphate 0.3g~2g/L, magnesium sulfate 0.2g~0.5g/L, carbon
Sour calcium 0.2g~2g/L, zinc sulfate 0g~0.4g/L, quick-acting carbon source glucoses 1g~10g/L, sodium chloride 2g~10g/L, sulfuric acid
0.0~0.6/L of manganese can influence the yield of the growth of thalline, the formation of gemma and parasporal crystal protein, and preliminary screening is several
The individual culture medium for comparatively facilitating thalli growth is combined, therefore this experiment is compared to the culture medium that preliminary screening goes out, and is
Culture medium further optimizes offer foundation.It is as follows for 4 kinds of basal mediums of examination：
No. 1：Cornstarch 20g/L, soybean cake powder 27g/L, dusty yeast 5.5g/L, dipotassium hydrogen phosphate 0.3g/L, magnesium sulfate
0.2g/L, calcium carbonate 0.4g/L, zinc sulfate 0.2g/L, glucose 1g/L, sodium chloride 2g/L, manganese sulfate 0.02g/L, pH7.0-
7.2。
No. 2：Peptone 1.2g/L, cornstarch 8.7g/L soybean cake powder 32g/L, dusty yeast 3g/L, potassium dihydrogen phosphate
0.7g/L, magnesium sulfate 0.3g/L, calcium carbonate 0.4g/L, zinc sulfate 0.2g/L, pH7.0-7.2.
No. 3：Peptone 2g/L, cornstarch 20g/L soybean cake powder 25g/L, dusty yeast 5g/L, potassium dihydrogen phosphate 0.3g/
L, magnesium sulfate 0.3g/L, calcium carbonate 1g/L, zinc sulfate 0.2g/L, glucose 1g/L, pH7.0-7.2.
LB culture mediums：Dusty yeast 5g/L, peptone 10g/L, sodium chloride 10g/L, pH7.0-7.2.
Above-mentioned 4 kinds of culture mediums are dissolved in distilled water in proportion, adjusts after pH, is fitted into 150mL conical flasks, each bottle
30mL, 121 DEG C of high pressure steam sterilizations 20 minutes.Using LB cultures 18h, thalline quantity 108Cfu/mL nutrient solutions as seed, its
It is 1% to be inoculated with inoculum concentration；25 DEG C, 150r/min shaken cultivations.Test 3 repetitions.Culture 24, sampling in 48 hours, sample respectively
Total viable cell is surveyed using direct 10 times of serial dilutions rubbing methods, heating is killed after vegetative cell using 10 times of graded series
Method of dilution butteron on plate surveys Number of spores.
Fig. 1 result of the tests show that No. 1 culture medium is in 48h and 72h, spore production highest.Follow-up medium optimization is 1
Carried out on number medium base.
Note：Capitalization represents the significance level of difference (p<0.01), when each processing below alphabetical identical not show
Write, it is just extremely notable when only letter is completely different；Similarly hereinafter.
The optimization of the bacillus thuringiensis bacterial strain B-Y7-1 culture mediums of embodiment 2
The culture medium based on No. 1 culture medium, using L9(34) orthogonal investigate cornstarch, soybean cake powder, ferment
Female powder, CaCO3Influence of the consumption to spore production, experimental design is shown in Table 1, prepares 9 kinds of fermentation mediums, in addition to inspection target,
The same basal medium of culture medium other compositions, medium sterilization, inoculation, fermentation, detection method be the same as Example 1.
The experimental factor of table 1 and level
Table 2：Medium culture optimizes orthogonal experiments
In fermenting and producing, it is desirable to obtain the gemma of sufficient amount, the medium optimization orthogonal test research of table 2 in a short time
As a result show, No. 7 processing combination (A3B1C3D2) spore production highests, wherein soybean cake powder consumption influences most on spore production
Greatly, next CaCO3Consumption；Possible optimum combination is A3B1C1D2.
Soybean cake powder consumption is further investigated, result of the test table 3 shows, soybean cake powder optimum dose is 25g/L.Through experiment
Verify (table 4), determine that optimal culture medium composition is soybean cake powder 25g/L, cornstarch 24g/L, dipotassium hydrogen phosphate 0.3g/L,
Magnesium sulfate 0.2g/L, zinc sulfate 0.2g/L, calcium carbonate 0.6g/L, dusty yeast 5.0g/L, glucose 1g/L, sodium chloride 2g/L, sulphur
Sour manganese 0.02g/L；48h spore productions are 1.86 × 1010cfu/mL。
Table 3：Spore production during various concentrations soybean cake powder fermentation 48h
Table 4：Best medium recipe verification
The optimization of the bacillus thuringiensis bacterial strain B-Y7-1 fermentation conditions of embodiment 3
Using Optimal Medium, using Shaking culture, research different initial pH, liquid amount, inoculum concentration, fermentation periods are to bud
The influence of spore yield, parasporal crystal protein.Under the conditions of liquid amount 1/5, the ﹪ of inoculum concentration 3, determine initial pH6.5,7,7.5,8 and be total to
Spore production under 4 gradients；Under the conditions of most suitable initial pH, the ﹪ of inoculum concentration 3, determine liquid amount be 1/5,1/3,1/2 when
Spore production；Under the conditions of most suitable initial pH, liquid amount, gemma production when inoculum concentration is respectively 1 ﹪, 3 ﹪, 5 ﹪, 7 ﹪ is determined
Amount；In most suitable initial, liquid amount, inoculum concentration condition, a gemma and parasporal crystal protein yield are determined every 8h.
Protein content is detected：2mL zymotic fluids 10000rpm is taken to centrifuge 10min, precipitation sterile water washing 2 times, use
0.5mol·L-1NaCl washings precipitation 2 times；Precipitation adds 1mL lysates (50mM Na2CO3, 10mM EDETATE SODIUMs, before use
Plus final concentration of 3% mercaptoethanol, 9.5) pH be adjusted to, and 5~6h is cracked at 4 DEG C；10000rpm centrifuges 10min, takes supernatant
Add 2mL 4M NaAc-HAc (pH=4.5), 4 DEG C of precipitation 4h.4 DEG C (10000rpm/20min) is centrifuged, and is precipitated with sterile washing
2mL pH 9.5 50mM Na are dissolved in after washing2CO3Solution, protein content is directly surveyed using ultramicron nucleic acid-protein detector.
Result of the test shows：Shake flask fermentation is most suitable to be initially pH 7.0 (Fig. 2), and liquid amount is 1/5 (Fig. 3), and inoculum concentration is
3% (Fig. 4).
Under optimum condition, different fermentations time gemma and parasporal crystal protein yield result are shown in Fig. 5 and Fig. 6.Difference hair
Ferment time spore production significant difference, 72h gemma amount highests, reaches 5.6 × 1010cfu/mL；Different fermentations time albumin crystal
Content has pole significant difference, 40h albumin crystal content highests of fermenting, and reaches 3.931mg/mL.Consider spore production, albumen
Crystal yield and production cost, fermentation period are advisable with 40h.
The bacillus thuringiensis bacterial strain B-Y7-1 ferment tank methods of embodiment 4
Prepare fermentation medium:Soybean cake powder 25g/L, cornstarch 24g/L, dipotassium hydrogen phosphate 0.3g/L, seven hydration sulphur
Sour magnesium 0.2g/L, zinc sulfate 0.2g/L, calcium carbonate 0.6g/L, dusty yeast 5.0g/L, glucose 1g/L, sodium chloride 2g/L, sulfuric acid
Manganese 0.02g/L, it is 7.2 to adjust initial pH.Add in air blow tank with machinery agitation, coefficient 75%, disappear 30min in fact；Adopt
With LB cultures 18h, thalline quantity 108Cfu/mL nutrient solutions are as seed, and it is 3% that it, which is inoculated with inoculum concentration,；250r/min, 27 DEG C of hairs
Ferment, froth breaking is automatically controlled by defoamer of soya-bean oil；Ventilation ratio is set to 1 in fermentation process:0.8 and 1:1.2, pH are set to
It is 7.0 and spontaneous fermentation (not controlling pH) to control pH, regulates and controls fermentation process.Timing sampling in fermentation process, determines Number of spores
And protein content.
Result of the test shows：Ventilation ratio 1:32 hours protein yield maximums of fermentation under the conditions of 1.2, in the control conditions of pH 7.0
Lower protein yield is 2.57mg/mL, hence it is evident that higher than the protein yield for not controlling pH spontaneous fermentation processes；Air agitation fermentation 32 is small
When gemma amount be more than 4 × 109Cfu/mL (table 5).Ventilation ratio 1:Air agitation is fermented under the conditions of 0.8, in the control conditions of pH 7.0
Protein yield is 2.24mg/mL at lower 28 hours, higher than the protein yield for not controlling pH spontaneous fermentation processes, and gemma amount is 2.4
×109cfu/mL；The pH spontaneous fermentations process gemma amount of 32 hours is not controlled to reach 1 × 109Cfu/mL (table 6).Consider bud
Spore yield and protein yield two indices, using air agitation liquid deep layer fermenting, 27 DEG C of fermentation temperature, suitable ventilation ratio is 1:
1.2, control ph is 7.0 in fermentation process, and fermentation time is 32 hours.
The ventilation ratio of table 5 is 1:The change of albumen and gemma amount in 1.2 fermentation process
The ventilation ratio of table 6 is 1:The change of albumen and gemma amount in 0.8 fermentation process
Claims (10)
1. a kind of fermentation medium, it is characterised in that the formula of described fermentation medium is as follows：
Soybean cake powder 21-31g/L, cornstarch 16-24g/L, calcium carbonate 0.4-0.8g/L, dusty yeast 3-7g/L, phosphoric acid hydrogen two
Potassium 0.3g/L, magnesium sulfate 0.2g/L, zinc sulfate 0.2g/L, glucose 1g/L, sodium chloride 2g/L, manganese sulfate 0.02g/L.
2. culture medium as claimed in claim 1, it is characterised in that described culture medium, it is soybean cake powder 25g/L that it, which is formulated,
Cornstarch 24g/L, dipotassium hydrogen phosphate 0.3g/L, magnesium sulfate 0.2g/L, zinc sulfate 0.2g/L, calcium carbonate 0.6g/L, dusty yeast
5.0g/L, glucose 1g/L, sodium chloride 2g/L, manganese sulfate 0.02g/L.
3. culture medium as claimed in claim 1 or 2, it is characterised in that the pH of described culture medium is 7.0.
4. application of the culture medium in bacillus thuringiensis B-Y7-1 strain fermentation cultures described in claim 1 or 2.
5. application as claimed in claim 4, it is characterised in that the preservation of described bacillus thuringiensis B-Y7-1 bacterial strains is compiled
Number be CCTCC No.M2011267.
6. a kind of method of fermented and cultured bacillus thuringiensis B-Y7-1 bacterial strains, it is characterised in that described method, is to use
Culture medium described in claim 1 or 2 cultivates bacterial strain.
7. method as claimed in claim 6, it is characterised in that as follows the step of described method：
Step 1) culture medium preparation：
Fermentation medium is prepared, it is 6.5-8.0 to adjust initial pH；
Step 2) packing：
The culture medium of preparation is loaded into triangular flask, liquid amount is the 1/5~1/2 of triangular flask dischargeable capacity；
Step 3) sterilizing：
121 DEG C of high pressure steam sterilizations 30 minutes；
Step 4) liquid spawn preparation：
The bacillus thuringiensis single bacterium colony of picking activation, is inoculated in LB fluid nutrient mediums, 28 DEG C of shaken cultivation 18h, thalline
Quantity reaches 108Cfu/mL, is used as seed liquor；
Step 5) inoculation：
According to 1%~7% inoculum concentration, sterile working is inoculated in step 3) sterilizing after culture medium in；
Step 6) fermentation：
It is placed in complete warm shaking table, 27 DEG C, 8~72h of 150r/min shaken cultivations.
8. method as claimed in claim 7, it is characterised in that the fermentation medium in described method adjusts initial pH and is
7.0, liquid amount for triangular flask dischargeable capacity 1/5,121 DEG C of high pressure steam sterilizations 30 minutes；Using LB cultures 18h, thalline number
Amount 108Cfu/mL nutrient solutions are as seed, and it is 3% that it, which is inoculated with inoculum concentration,；27 DEG C, 150r/min shaken cultivations, ferment 40h albumen
Crystalline content highest, reaches 3.931mg/mL, and Number of spores is more than 1 × 1010cfu/mL。
9. method as claimed in claim 6, it is characterised in that as follows the step of described method：
Step 1) culture medium preparation：
Fermentation medium is prepared, it is 7.2 to adjust initial pH；
Step 2) sterilizing：
Fermentation medium is added in air blow tank with machinery agitation, coefficient 75%, 121 DEG C, the real 30min that disappears；
Step 3) inoculation：
By thalline quantity 108Cfu/mL seed culture fluids, by 3% inoculum concentration, are inoculated into fermentation tank；
Step 4) fermentation：
Ventilation ratio 1:0.8~1:1.2,250r/min, 27 DEG C of fermentations, froth breaking, fermentation process are automatically controlled by defoamer of soya-bean oil
Middle pH is natural or controls pH to be 7.0, fermentation time 8-72 hours.
10. method as claimed in claim 9, it is characterised in that the fermentation medium in described method, adjusting initial pH is
7.2, add in air blow tank with machinery agitation, coefficient 75%, disappear 30min in fact；Using LB cultures 18h, thalline quantity
108Cfu/mL nutrient solutions are as seed, and it is 3% that it, which is inoculated with inoculum concentration,；Ventilation ratio 1:1.2,250r/min, 27 DEG C of fermentations, with beans
Oil is that defoamer automatically controls and controls pH to be 7.0 in froth breaking, fermentation process, and fermentation time is 32 hours, and protein yield is
2.57mg/mL, gemma amount is more than 4 × 109cfu/mL。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710420533.9A CN107090420B (en) | 2017-06-07 | 2017-06-07 | Fermentation culture method of bacillus thuringiensis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710420533.9A CN107090420B (en) | 2017-06-07 | 2017-06-07 | Fermentation culture method of bacillus thuringiensis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107090420A true CN107090420A (en) | 2017-08-25 |
| CN107090420B CN107090420B (en) | 2021-01-26 |
Family
ID=59640015
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710420533.9A Active CN107090420B (en) | 2017-06-07 | 2017-06-07 | Fermentation culture method of bacillus thuringiensis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107090420B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441454A (en) * | 2018-05-16 | 2018-08-24 | 广西青又青生物肥业有限公司 | A kind of fluid nutrient medium and cultivation and fermentation method of bacillus thuringiensis |
| CN109652342A (en) * | 2019-02-01 | 2019-04-19 | 福建农林大学 | A method of Bt liquid fermentation medium is prepared by raw material of vegetable castoff |
| CN110396490A (en) * | 2019-08-15 | 2019-11-01 | 江苏苏滨生物农化有限公司 | A method of culture Brevibacillus brevis |
| CN112501083A (en) * | 2020-12-22 | 2021-03-16 | 武汉科诺生物科技股份有限公司 | Bacillus thuringiensis liquid fermentation medium and fermentation culture method |
| 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 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR94103E (en) * | 1963-03-05 | 1969-07-04 | Zaidan Hojin Biseibutsu | Process for the preparation of bleomycin. |
| CN1246284A (en) * | 1999-09-14 | 2000-03-08 | 中国科学院生态环境研究中心 | Process for preparing microbe insecticide with high-concentration waste organic water in fermentation industry |
| CN103160450A (en) * | 2011-12-15 | 2013-06-19 | 河北农业大学 | Broad-spectrum efficient novel bacillus thuringiensis strain, microbial inoculum and application thereof |
| CN103627651A (en) * | 2013-10-08 | 2014-03-12 | 暨南大学 | Bacillus thuringiensis for decomposing zearalenone and application thereof |
| CN103992968A (en) * | 2014-04-22 | 2014-08-20 | 安徽清明环保科技有限公司 | Household garbage deodorizing and sterilizing composite inoculant and preparation method thereof |
| CN104336076A (en) * | 2014-08-29 | 2015-02-11 | 湖北省生物农药工程研究中心 | Preparation process of live bacillus thuringiensis preparation |
| CN104357358A (en) * | 2014-11-11 | 2015-02-18 | 山东苏柯汉生物工程股份有限公司 | Compound bacterium for preventing and treating root-knot nematode and preparation method thereof |
| CN104789226A (en) * | 2015-03-13 | 2015-07-22 | 迪斯科化工集团股份有限公司 | Charcoal-based microbial soil conditioner and preparation method thereof |
-
2017
- 2017-06-07 CN CN201710420533.9A patent/CN107090420B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR94103E (en) * | 1963-03-05 | 1969-07-04 | Zaidan Hojin Biseibutsu | Process for the preparation of bleomycin. |
| CN1246284A (en) * | 1999-09-14 | 2000-03-08 | 中国科学院生态环境研究中心 | Process for preparing microbe insecticide with high-concentration waste organic water in fermentation industry |
| CN103160450A (en) * | 2011-12-15 | 2013-06-19 | 河北农业大学 | Broad-spectrum efficient novel bacillus thuringiensis strain, microbial inoculum and application thereof |
| CN103627651A (en) * | 2013-10-08 | 2014-03-12 | 暨南大学 | Bacillus thuringiensis for decomposing zearalenone and application thereof |
| CN103992968A (en) * | 2014-04-22 | 2014-08-20 | 安徽清明环保科技有限公司 | Household garbage deodorizing and sterilizing composite inoculant and preparation method thereof |
| CN104336076A (en) * | 2014-08-29 | 2015-02-11 | 湖北省生物农药工程研究中心 | Preparation process of live bacillus thuringiensis preparation |
| CN104357358A (en) * | 2014-11-11 | 2015-02-18 | 山东苏柯汉生物工程股份有限公司 | Compound bacterium for preventing and treating root-knot nematode and preparation method thereof |
| CN104789226A (en) * | 2015-03-13 | 2015-07-22 | 迪斯科化工集团股份有限公司 | Charcoal-based microbial soil conditioner and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 杨梅 等: "苏云金芽孢杆菌LLB19发酵培养基的优化", 《福建师范大学学报(自然科学版)》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441454A (en) * | 2018-05-16 | 2018-08-24 | 广西青又青生物肥业有限公司 | A kind of fluid nutrient medium and cultivation and fermentation method of bacillus thuringiensis |
| CN109652342A (en) * | 2019-02-01 | 2019-04-19 | 福建农林大学 | A method of Bt liquid fermentation medium is prepared by raw material of vegetable castoff |
| CN110396490A (en) * | 2019-08-15 | 2019-11-01 | 江苏苏滨生物农化有限公司 | A method of culture Brevibacillus brevis |
| CN110396490B (en) * | 2019-08-15 | 2020-10-09 | 江苏苏滨生物农化有限公司 | Method for culturing brevibacillus brevis |
| CN112501083A (en) * | 2020-12-22 | 2021-03-16 | 武汉科诺生物科技股份有限公司 | Bacillus thuringiensis liquid fermentation medium and fermentation culture method |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107090420B (en) | 2021-01-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107090420A (en) | A kind of fermentation culture method of bacillus thuringiensis | |
| CN103789234B (en) | A kind of bacillus amyloliquefaciens and application thereof | |
| CN103627662B (en) | A kind of Bradyrhizobium sp Arachis and uses thereof | |
| CN101845409B (en) | Bacillus firmus and application thereof | |
| CN103160442B (en) | Paecilomyceslilacinus strain having strong pathogenicity for diaphorina citri | |
| CN105861376B (en) | A kind of serratia marcescens and its separation method and application | |
| CN105420115B (en) | It is a kind of for halimasch spore separation, culture culture medium and methods and applications | |
| CN101654658B (en) | Pesticidal metarhizium anisopliae strain | |
| CN110423712A (en) | A kind of auspicious Russia's series bacillus of Pierre and its application | |
| CN107502581A (en) | One plant of Serratia bacterial strain | |
| CN106676049A (en) | Bacillus amyloliquefaciens strain and application thereof | |
| CN104726378B (en) | The method for improving salt stress turfgrass defence enzyme activity using Salt-tolerant microbial agent is strengthened | |
| CN106834130A (en) | One plant of Strain of Beauveria bassiana and its application to grub larva highly pathogenicity | |
| CN112410261B (en) | Bacillus siamensis MC2-1 and application thereof | |
| CN103243030B (en) | Lecanicilliumpsalliotae strain used for preventing and treating diaphorina citri | |
| CN107384823A (en) | One plant of grease degrading strain and its application | |
| CN105767009A (en) | Production method of nonpathogenic fusarium oxysporum-containing plant vaccine preparation | |
| CN109769858B (en) | Plant root-knot nematode inhibitor and application thereof | |
| CN102329760B (en) | New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof | |
| CN104560740B (en) | Metarhizium anisopliae and its application of one plant of preventing and treating oil tea as larva | |
| CN106399180A (en) | Acetochlor herbicide degrading bacteria and production method and use of agent of acetochlor herbicide degrading bacteria | |
| CN109169712A (en) | A kind of compound biocontrol fungicide and its preparation method and application | |
| CN108913625A (en) | Salt tolerant streptomycete, its microbial inoculum and its microbial inoculum are promoting the application in plant growth | |
| CN106520635B (en) | One plant of bacillus cereus NJSZ-13 and its application | |
| CN107629985A (en) | One plant of endophytic bacterium for having antagonism to plant pathogenic fungi |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |