CN112501083A - Bacillus thuringiensis liquid fermentation medium and fermentation culture method - Google Patents
Bacillus thuringiensis liquid fermentation medium and fermentation culture method Download PDFInfo
- Publication number
- CN112501083A CN112501083A CN202011525927.9A CN202011525927A CN112501083A CN 112501083 A CN112501083 A CN 112501083A CN 202011525927 A CN202011525927 A CN 202011525927A CN 112501083 A CN112501083 A CN 112501083A
- Authority
- CN
- China
- Prior art keywords
- bacillus thuringiensis
- fermentation
- culture medium
- culture
- strain
- 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
Images
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
-
- 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
- C12N1/04—Preserving or maintaining viable microorganisms
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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 invention discloses a liquid fermentation culture method of bacillus thuringiensis JQ23 for preventing and treating bradysia odoriphaga, which uses a fermentation culture medium formula comprising 30-50g/L of glucose, 25-35g/L of soybean cake powder, 5-15g/L of peanut meal, 10-30g/L of peptone, 2-5g/L of yeast powder, 0.2-0.5g/L of monopotassium phosphate, 0.2-0.5g/L of ammonium sulfate, 0.1-0.3g/L of calcium chloride, 0.01-0.05g/L of manganese chloride and the balance of sterile water. The spore content of the bacillus thuringiensis cultured by the invention can reach 8.25 multiplied by 109cfu/mL, the corrected mortality rate of the 2-year-old Chinese chive maggot larvae for 3 days can reach 93%, the content of infectious microbes is low, and the suspending agent prepared by the fermentation liquor is stable in placement performance at normal temperature and is convenient for long-distance transportation and long-term storage.
Description
Technical Field
The invention belongs to the field of biocontrol, and particularly relates to a bacillus thuringiensis liquid fermentation culture medium and a fermentation culture method of bacillus thuringiensis.
Background
Bradysia odoriphaga belongs to the diptera muscomyiidae, which is also called yellow foot muscae, mainly harms vegetables of the liliaceae, such as Chinese chives, green Chinese onions, green Chinese onions, garlic and the like, occasionally also harms lettuce, green vegetables, celery and the like, the larvae have the greatest harm to the vegetables, the vegetables mostly live on the surface layer of soil, and bulbs and tender stems which are clustered under the Chinese chives are damaged by eating, so that the young stems are rotten, and the Chinese chives are withered and yellow to die. In recent years, under the influence of factors such as cultivation conditions and pest resistance, Chinese chive maggots erupt in a large scale in partial areas, and great economic loss is caused to vegetable farmers. At present, methods for preventing and treating Chinese chive maggots mainly comprise a material method and a chemical method, and due to the defects that physical prevention and treatment methods (sugar-vinegar liquid, ozone and high-temperature films) are complicated to operate and the prevention and treatment effect is greatly influenced by the environment, most vegetable growers choose chemical means such as chemical pesticide root irrigation to prevent and treat Chinese chive maggots, but because Chinese chive maggots are fast in propagation, high in concealment and high in prevention and treatment difficulty, the phenomenon that pesticide residues exceed the standard due to the fact that the vegetable growers use chemical pesticides irregularly is caused, the environment is polluted, and meanwhile the health of human beings and other animals is harmed.
Bacillus thuringiensis (Bt) has been studied extensively by researchers as a microorganism that produces endotoxins (parasporal crystals) and exotoxins. At present, the microorganism has a certain prevention and treatment effect on various plant diseases and insect pests, and does not pollute the environment and influence human health, so that various bacillus thuringiensis related preparations are developed successively, and the market is sold by more than 100 at present. However, research and application of bacillus thuringiensis preparations for preventing and treating Chinese chive maggots are few, for example, the effect of preventing and treating Chinese chive maggots of bacillus thuringiensis screened in academic america can reach about 75%, and the effect of preventing and treating Chinese chive maggots of bacillus thuringiensis screened in vitamin solving can reach about 84.85%, but at present, the bacillus thuringiensis preparations are still in a research stage, and no product is formed. Therefore, the development of the bacillus thuringiensis product with certain control effect on the Chinese chive maggots has important significance for guaranteeing food safety, protecting ecological environment and promoting agricultural economic development.
In order to develop a biological agent for preventing and treating Chinese chive maggots, the inventor introduces a Bacillus thuringiensis JQ23 strain which has certain control effect on Chinese chive maggots and is described in CN104099277A by Wuhan Keno biotech Co., Ltd, the rule of formation of total spores and crystal protein is not clear in the process of culturing the Bacillus thuringiensis JQ23 at present, the level and biological potency of the spores produced by fermentation are not high, and the scale production, development and utilization of the Bacillus thuringiensis JQ23 strain are restricted. Therefore, a fermentation culture technology suitable for the strain is developed, a large number of spore or semi-spore crystals are obtained, the development and utilization of related preparations of the strain can be effectively promoted, and the method has important significance for the control of bradysia odoriphaga.
Disclosure of Invention
The invention aims to provide a liquid fermentation culture medium and a fermentation culture method aiming at the strain, aiming at the defects of low fermentation level, unstable product quality and the like of the prior bacillus thuringiensis.
A Bacillus thuringiensis liquid fermentation culture medium comprises the following formula: 30-50g/L of glucose, 25-35g/L of soybean cake powder, 5-15g/L of peanut meal, 10-30g/L of peptone, 2-5g/L of yeast powder, 0.2-0.5g/L of monopotassium phosphate, 0.2-0.5g/L of ammonium sulfate, 0.1-0.3g/L of calcium chloride, 0.01-0.05g/L of manganese chloride and the balance of sterile water.
Preferably, the formulation of the medium is as follows: 40g/L of glucose, 30g/L of soybean cake powder, 15g/L of peanut meal, 30g/L of peptone, 5g/L of yeast powder, 0.3g/L of monopotassium phosphate, 0.3g/L of ammonium sulfate, 0.2g/L of calcium chloride, 0.05g/L of manganese chloride and the balance of sterile water.
A fermentation culture method of Bacillus thuringiensis comprises the step of carrying out fermentation culture on Bacillus thuringiensis seed liquid by using the culture medium.
One of the methods is suitable for small-scale culture, and the method is a shake flask culture method, and comprises the following specific steps: adjusting the initial pH of the culture medium to 7.0-7.2, inoculating the Bacillus thuringiensis seed solution after sterilization, wherein the inoculation amount is 1-5% (by weight), placing the inoculated shake flask in a constant temperature shaking table, and carrying out shaking culture at 28-32 ℃ and 320rpm for 40-48 h. After the culture by the method, the spore shedding rate can reach more than 85 percent, and the number of spores can reach 8.25 multiplied by 109The cfu/mL, diluted 100 times, can reach 93% for 3 days of corrected mortality of 2-instar larvae.
One of the culture methods suitable for large-scale production is a fermentation tank culture method, and specifically comprises the following steps: adjusting the initial pH of the culture medium to 7.0-7.2, sterilizing, inoculating Bacillus thuringiensis seed solution with an inoculation amount of 1-5%, setting the initial rotation speed of a fermentation tank to 300rpm, controlling the rotation speed to be associated with 40% DO in the fermentation process, and controlling the aeration ratio of 0-6h to be 1: 0.8; the ventilation ratio of 6-30h is 1: 1.2; the aeration ratio from 30h to the end of fermentation is 1:0.8, the tank pressure is maintained at 0.05-0.06Mpa, and the fermentation period is 40-48 h. After the method is adopted for culture, the number of spores can reach 7.50 multiplied by 109cfu/mL, 100-fold dilution, achieved 83% corrected mortality for 2 instar larvae for 3 days.
Preferably, the thallus concentration of the bacillus thuringiensis seed liquid is 108cfu/mL, activating the bacillus thuringiensis strain at normal temperature, inoculating the activated bacillus thuringiensis strain into a slant culture medium under an aseptic condition, and culturing for 72-96h in an incubator at 30 ℃ to obtain a bacillus thuringiensis strain slant; washing the prepared strain slant with 50mL of sterile water, performing water bath at 65 ℃ for 30min, inoculating to LB liquid culture medium according to 1% of inoculum size, and culturing at 220rpm for 12-15h to obtain the strainIs a seed liquid.
A preparation method of a bacillus thuringiensis suspending agent comprises the following steps: acidifying the Bacillus thuringiensis fermentation liquor to pH4.0-4.5, sieving with 100 mesh sieve, collecting filtrate, adding 3-5 ‰ sodium benzoate, adding 1-3 ‰ xanthan gum to adjust the viscosity of the fermentation liquor to 200-2And (6) subpackaging.
The bacillus thuringiensis is bacillus thuringiensis JQ23 which is disclosed in the patent application with the application number of 201410338466.2, the publication number of CN104099277A and the strain preservation number of CGMCC No. 9375.
The invention has the following beneficial effects:
(1) the optimized culture medium and culture conditions provided by the invention are adopted to carry out shake flask and fermentation tank fermentation tests on the strain JQ23, the number of spores and the indoor biological value are obviously improved compared with the fermentation level of the same strain in CN104099277A, and the culture medium provided by the invention has the advantages of simple components, convenient preparation, low cost, controllable fermentation conditions, simplicity, feasibility and short fermentation period.
(2) The suspending agent prepared by the fermentation method provided by the invention has good dispersibility in water, and stable spore number and indoor biological value after being stored for 12 months at normal temperature, and is convenient for long-distance transportation and long-term storage.
Drawings
FIG. 1: the spore yield of the bacillus thuringiensis fermented by different culture media.
FIG. 2: spore yield analysis plots for different initial pH.
FIG. 3: spore yield analysis chart of different inoculum sizes.
FIG. 4: spore yield analysis chart at different rotating speeds.
The capital letters A, B, C, D in the figure indicate significant differences (p <0.05) when the letters are different and insignificant differences (p >0.05) when the letters are the same.
Detailed Description
The present invention will be described in detail below with reference to specific examples.
The bacillus thuringiensis JQ23 strain is obtained by the transfer of plant protection research institute of academy of sciences of agriculture and forestry, Hebei, and has been preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.9375 in 6-24 th 2014, and has been disclosed in the patent application with the application number of 201410338466.2, the publication number of CN104099277A, 10-15 th 2014, and the application does not relate to the preservation of biological materials.
EXAMPLE 1 screening of liquid fermentation Medium
In early fermentation test research, the applicant finds that 20-40g/L of corn starch as a slow-acting carbon source, 30-50g/L of glucose as a fast-acting carbon source, 15-35g/L of soybean cake powder as a slow-acting nitrogen source, 5-20g/L of peanut meal, 10-15g/L of corn flour, 5-10g/L of yeast powder, 10-30g/L of peptone as a fast-acting nitrogen source, 0.2-0.5g/L of inorganic salt monopotassium phosphate, 0.2-0.5g/L of ammonium sulfate, 0.1-0.3g/L of calcium chloride and 0.01-0.05g/L of manganese chloride can influence the growth of thalli, the formation of spores and the generation of parasporal crystals, screens a plurality of culture mediums which are favorable for the growth of thalli to combine for primary fermentation and shake flask culture, and compares the culture with the culture mediums provided by CN104099277A, provide basis for further optimization of the culture medium, and the 4 fermentation culture media tested are as follows:
medium No. 1: 27g/L of corn starch, 16g/L of soybean cake powder, 5g/L of peanut meal, 10g/L of corn flour, 14g/L of peptone, 4g/L of yeast powder, 8g/L of bran, 0.25g/L of ammonium sulfate, 0.05g/L of manganese chloride, the balance of sterile water and the pH value of 7.0-7.2.
Medium No. 2: 30g/L of corn starch, 25g/L of soybean cake powder, 15g/L of peanut meal, 20g/L of peptone, 5g/L of yeast powder, 0.3g/L of monopotassium phosphate, 0.3g/L of ammonium sulfate, 0.2g/L of calcium chloride, 0.05g/L of manganese chloride, the balance of sterile water and pH of 7.0-7.2.
Medium No. 3: 40g/L of glucose, 30g/L of soybean cake powder, 10g/L of peanut meal, 30g/L of peptone, 5g/L of yeast powder, 0.3g/L of monopotassium phosphate, 0.3g/L of ammonium sulfate, 0.2g/L of calcium chloride, 0.05g/L of manganese chloride, the balance of sterile water and pH of 7.0-7.2.
Control medium: 15g/L of corn starch, 25g/L of soybean cake powder, 12g/L of cottonseed cake powder, 5g/L of yeast powder, 0.3g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate and the balance of sterile water, wherein the pH value is 7.0-7.2.
Dissolving the raw materials of the culture media in distilled water according to a ratio, adjusting the pH of the culture media to 7.0, subpackaging the mixture into 500mL conical flasks according to the liquid loading amount of 30mL, wherein the glucose is independently sterilized at 118 ℃ for 30min, the other materials are sterilized at 121 ℃ for 30min, inoculating the seed solution according to the inoculation amount of 1% when the materials are cooled to 40 ℃, performing shaking culture at 30 ℃ and 280rpm, repeating the test for 3 times, and sampling after 48 hours to determine the spore number and the biological titer of the fermentation liquid. Wherein the spore number is measured by adopting a gradient dilution coating method, and the diluted sample is subjected to water bath at 70 ℃ for 20min before coating; the indoor biological potency measurement method refers to patent CN 104099277A.
The preparation method of the seed liquid comprises the following steps: activating the strain of Bacillus thuringiensis JQ23 at normal temperature, inoculating the strain into a slant culture medium under an aseptic condition, and culturing for 72-96h in an incubator at 30 ℃ to obtain a Bacillus thuringiensis slant; washing the prepared strain slant with 50mL of sterile water, performing water bath at 65 deg.C for 30min, inoculating to LB liquid culture medium according to 1% inoculum size, culturing at 220rpm for 12-15h to obtain seed solution with thallus concentration of 108cfu/mL。
FIG. 1 shows that the number of spores obtained by fermentation using culture medium No. 3 is up to 7.20X 109cfu/mL, and the culture medium optimization in the later period is carried out on the basis of the culture medium No. 3.
EXAMPLE 2 optimization of liquid fermentation Medium
Based on the No. 3 medium, L9 (3) was used4) The influence of the dosage of glucose, soybean cake powder, peanut meal and ammonium sulfate on the yield of spores is examined through orthogonal test design, the test design is shown in table 1, 9 fermentation media are prepared, other components of the media are the same as basic media except for the examined indexes, and the media sterilization, inoculation, fermentation and detection methods are the same as those in example 1.
TABLE 1 test factors and levels
Table 2: culture medium optimization orthogonal test results
In fermentation production, it is desirable to obtain a sufficient number of spores in the shortest time, and the results of the culture medium optimization orthogonal test in Table 2 show that the spore yield is the highest for treatment combination No. 5 (A2B2C3D1), where the amount of glucose has the greatest effect on spore yield, followed by the soybean meal; the best possible combination is (A2B2C3D2) it was verified that when the formula A2B2C3D2 was used as the fermentation medium, the spore count reached 7.85X 109cfu/mL。
Example 3 optimization of Shake flask fermentation conditions
Carrying out shake flask culture by using an optimized culture medium, researching the influence of different initial pH values, inoculation amounts and rotating speeds of the culture medium on spore yield and biological titer, uniformly carrying out shake flask test by using a 500mL conical flask according to a 30mL liquid loading amount, and measuring the spore yield under 5 gradients of initial pH5.5, 6.0, 6.5, 7.0 and 7.5 under the condition that the inoculation amount is 1%; under the condition of the optimal initial pH, measuring the spore yield when the inoculation amount is respectively 1%, 3%, 5% and 7%; under the conditions most suitable for the initial pH and the inoculum size, the spore yields at 260rpm, 280rpm, 300rpm, and 320rpm, respectively, were determined. And performing a shake flask test by adopting an optimized culture medium under the conditions of optimal initial pH, inoculation amount and rotation speed, repeating the shake flask test according to the culture conditions provided in the patent CN104099277A, and comparatively inspecting the spore and biological titer levels of the fermentation sample.
The experimental results showed that the shaking flask fermentation medium had an optimum initial pH of 7.0 (FIG. 2), an inoculum size of 3% (FIG. 3) and a rotation speed of 300rpm (FIG. 4).
The spore level of the fermentation liquor obtained according to the culture conditions described in the literature is 5.58 multiplied by 108cfu/mL, the corrected mortality rate of the 2 nd larva can reach 75.67% after 100 times dilution, and the spore level of the fermentation liquid obtained by adopting the optimized culture condition is 8.25 multiplied by 109cfu/mL, the corrected mortality rate of the 2 nd larva by 100 times dilution can reach 93%, and the spore and biological potency levels are obviously improved (p) before optimization<0.05)。
Example 4 fermentation method of Bacillus thuringiensis in a 15L fully automatic fermentation tank
Performing fermentation tests on a full-automatic fermentation tank for more than 5 times by adopting a screened optimal formula, determining an optimal fermentation process, controlling the liquid loading of a culture medium to 50-60%, adjusting the initial pH to 7.0, wherein glucose is 0.08MPa and is independently sterilized and supplemented at 118 ℃, other materials are 0.1MPa and are sterilized at 121 ℃ for 30min, the inoculation amount is 3%, the culture temperature is 30 ℃, the initial rotation speed is controlled to be 300rpm, the rotation speed is related to 40% DO in the fermentation process, the time is 0-6h, and the aeration ratio is 1: 0.8; 6-30h, wherein the aeration ratio is 1: 1.2; 30 h-fermentation is finished, the aeration ratio is 1:0.8, the tank pressure is maintained at 0.05-0.06Mpa, the fermentation period is about 42h, and the fermentation liquid is acidified to pH4.2 after the fermentation is finished.
Test results show that when the technological parameters are adopted, the fermentation period can be shortened to 42 hours, the spore shedding rate reaches more than 85 percent, the viscosity of the put-in-tank acidified sample is 24.5CPS, and the spore number can reach 7.50 multiplied by 109cfu/mL, the corrected mortality rate of the 2-year-old Chinese chive maggot larvae for 3 days after 100-time dilution can reach 83%.
EXAMPLE 5 preparation and ambient storage of the suspending agent
Filtering an acidified sample obtained by fermenting a newly screened fermentation formula in a 15L fermentation tank by using a 100-mesh screen, taking a filtrate, adding 4 per thousand of sodium benzoate according to the mass ratio, adding 2 per thousand of xanthan gum to adjust the viscosity of the fermentation liquor to 200 per thousand of 300CPS to prepare a suspending agent, filling N2Then subpackaging, placing in (22 + -2) deg.C incubator, periodically sampling to determine spore number and indoor biological value, with a period of 1 year, and comparing with suspending agent prepared by fermentation of culture medium provided by patent CN104099277A, the results are shown in Table 3:
TABLE 3 spore and indoor bioassay horizontal stability of the suspension after 1 year of storage at Normal temperature
According to the test results, compared with the suspending agent prepared by the fermentation of the culture medium provided by CN104099277A, the suspending agent prepared by the fermentation of the culture medium provided by the invention has more stable spores and indoor bioassay levels after being placed for 1 year at normal temperature, and is convenient for long-term storage and transportation.
Claims (10)
1. A fermentation culture method of Bacillus thuringiensis is characterized by comprising the step of carrying out fermentation culture on Bacillus thuringiensis seed liquid by using a culture medium, wherein the formula of the culture medium is as follows: 30-50g/L of glucose, 25-35g/L of soybean cake powder, 5-15g/L of peanut meal, 10-30g/L of peptone, 2-5g/L of yeast powder, 0.2-0.5g/L of monopotassium phosphate, 0.2-0.5g/L of ammonium sulfate, 0.1-0.3g/L of calcium chloride, 0.01-0.05g/L of manganese chloride and the balance of sterile water.
2. The fermentation culture method of claim 1, wherein the formula of the culture medium is as follows: 40g/L of glucose, 30g/L of soybean cake powder, 15g/L of peanut meal, 30g/L of peptone, 5g/L of yeast powder, 0.3g/L of monopotassium phosphate, 0.3g/L of ammonium sulfate, 0.2g/L of calcium chloride, 0.05g/L of manganese chloride and the balance of sterile water.
3. The fermentative cultivation process according to claim 1 or 2, characterized in that it is a shake flask cultivation process, in particular as follows: adjusting the initial pH of the culture medium to 7.0-7.2, inoculating the Bacillus thuringiensis seed solution after sterilization with the inoculation amount of 1-5%, placing the inoculated shake flask in a constant temperature shaking table, and carrying out shaking culture at 28-32 ℃ and 260-320rpm for 40-48 h.
4. The fermentative cultivation process according to claim 1 or 2, characterized in that it is a fermenter cultivation process, in particular as follows: adjusting the initial pH of the culture medium to 7.0-7.2, inoculating the Bacillus thuringiensis seed solution after sterilization, wherein the inoculation amount is 1-5%, the initial rotation speed of a fermentation tank is set to be 300rpm, the control rotation speed is related to 40% DO in the fermentation process, and the aeration ratio is 1:0.8 after 0-6 h; the ventilation ratio of 6-30h is 1: 1.2; the aeration ratio from 30h to the end of fermentation is 1:0.8, the tank pressure is maintained at 0.05-0.06Mpa, and the fermentation period is 40-48 h.
5. The fermentation culture method according to claim 1 or 2, characterized in that: the Bacillus thuringiensis speciesThe bacterial concentration of the seed liquid was 108cfu/mL, activating the bacillus thuringiensis strain at normal temperature, inoculating the activated bacillus thuringiensis strain into a slant culture medium under an aseptic condition, and culturing for 72-96h in an incubator at 30 ℃ to obtain a bacillus thuringiensis strain slant; washing the prepared strain slant with 50mL of sterile water, carrying out water bath at 65 ℃ for 30min, then inoculating the strain into an LB liquid culture medium according to the inoculum size of 1%, and culturing at 220rpm for 12-15h to obtain the seed solution.
6. The fermentation culture method according to claim 1 or 2, wherein the bacillus thuringiensis is bacillus thuringiensis JQ23 with the strain preservation number of cgmcc.no. 9375.
7. A Bacillus thuringiensis liquid fermentation culture medium is characterized in that the formula of the culture medium is as follows: 30-50g/L of glucose, 25-35g/L of soybean cake powder, 5-15g/L of peanut meal, 10-30g/L of peptone, 2-5g/L of yeast powder, 0.2-0.5g/L of monopotassium phosphate, 0.2-0.5g/L of ammonium sulfate, 0.1-0.3g/L of calcium chloride, 0.01-0.05g/L of manganese chloride and the balance of sterile water.
8. Use of the medium of claim 7 in the fermentative culture of bacillus thuringiensis.
9. A Bacillus thuringiensis suspending agent is characterized in that: comprises the Bacillus thuringiensis fermentation broth acidified to pH4.0-4.5 obtained by the fermentation culture method according to claim 1, sodium benzoate and xanthan gum.
10. A method of preparing a bacillus thuringiensis suspension concentrate as claimed in claim 9, wherein: acidifying the Bacillus thuringiensis fermentation liquid obtained by the fermentation culture method according to claim 1 to pH4.0-4.5, sieving, taking the filtrate, adding 3-5 per mill of sodium benzoate according to the mass ratio, and then adding 1-3 per mill of xanthan gum to adjust the viscosity of the fermentation liquid to 200-300 CPS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011525927.9A CN112501083B (en) | 2020-12-22 | 2020-12-22 | Bacillus thuringiensis liquid fermentation culture medium and fermentation culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011525927.9A CN112501083B (en) | 2020-12-22 | 2020-12-22 | Bacillus thuringiensis liquid fermentation culture medium and fermentation culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112501083A true CN112501083A (en) | 2021-03-16 |
| CN112501083B CN112501083B (en) | 2023-01-03 |
Family
ID=74923097
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011525927.9A Active CN112501083B (en) | 2020-12-22 | 2020-12-22 | Bacillus thuringiensis liquid fermentation culture medium and fermentation culture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112501083B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114591851A (en) * | 2021-12-31 | 2022-06-07 | 广东省科学院化工研究所 | Agricultural microbial agent and preparation method thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1876807A (en) * | 2006-07-07 | 2006-12-13 | 中国科学院武汉病毒研究所 | Anti-locust bacillus thuringiensis and preparation method for Bt locust killing agent |
| CN101481668A (en) * | 2009-01-15 | 2009-07-15 | 山东省科学院中日友好生物技术研究中心 | Bacillus thuringiensis suspending agent for killing cotton spider mites, preparation and use |
| CN101669523A (en) * | 2009-08-19 | 2010-03-17 | 湖北省生物农药工程研究中心 | Production process of bacillus thuringiensis suspension concentrates |
| CN104099277A (en) * | 2014-07-16 | 2014-10-15 | 河北省农林科学院植物保护研究所 | Bacillus thuringiensis for preventing and controlling bradysia odoriphaga, and application of bacillus thuringiensis |
| CN105199986A (en) * | 2015-09-25 | 2015-12-30 | 武汉科诺生物科技股份有限公司 | Fed-batch fermentation culture method for bacillus thuringiensis engineering bacteria G033A |
| CN105624060A (en) * | 2015-12-30 | 2016-06-01 | 胡丽 | Thuringiensis bacillus fermentation medium |
| CN105907661A (en) * | 2016-04-08 | 2016-08-31 | 中国农业大学 | Industrial fermentation method for Bacillus subtillis |
| CN107090420A (en) * | 2017-06-07 | 2017-08-25 | 青岛农业大学 | A kind of fermentation culture method of bacillus thuringiensis |
| CN107912448A (en) * | 2017-12-04 | 2018-04-17 | 青岛农业大学 | Kill grub class pest bacillus thuringiensis micro-capsule suspension and preparation method |
| 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 |
-
2020
- 2020-12-22 CN CN202011525927.9A patent/CN112501083B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1876807A (en) * | 2006-07-07 | 2006-12-13 | 中国科学院武汉病毒研究所 | Anti-locust bacillus thuringiensis and preparation method for Bt locust killing agent |
| CN101481668A (en) * | 2009-01-15 | 2009-07-15 | 山东省科学院中日友好生物技术研究中心 | Bacillus thuringiensis suspending agent for killing cotton spider mites, preparation and use |
| CN101669523A (en) * | 2009-08-19 | 2010-03-17 | 湖北省生物农药工程研究中心 | Production process of bacillus thuringiensis suspension concentrates |
| CN104099277A (en) * | 2014-07-16 | 2014-10-15 | 河北省农林科学院植物保护研究所 | Bacillus thuringiensis for preventing and controlling bradysia odoriphaga, and application of bacillus thuringiensis |
| CN105199986A (en) * | 2015-09-25 | 2015-12-30 | 武汉科诺生物科技股份有限公司 | Fed-batch fermentation culture method for bacillus thuringiensis engineering bacteria G033A |
| CN105624060A (en) * | 2015-12-30 | 2016-06-01 | 胡丽 | Thuringiensis bacillus fermentation medium |
| CN105907661A (en) * | 2016-04-08 | 2016-08-31 | 中国农业大学 | Industrial fermentation method for Bacillus subtillis |
| CN107090420A (en) * | 2017-06-07 | 2017-08-25 | 青岛农业大学 | A kind of fermentation culture method of bacillus thuringiensis |
| CN107912448A (en) * | 2017-12-04 | 2018-04-17 | 青岛农业大学 | Kill grub class pest bacillus thuringiensis micro-capsule suspension and preparation method |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| FABRÍZIO SIQUEIRA BONIOLO等: "Oxygen supply in Bacillus thuringiensis fermentations: bringing new insights on their impact on sporulation and delta-endotoxin production", 《APPL MICROBIOL BIOTECHNOL》 * |
| 史应武、赵思峰、李国英: "新疆苏云金杆菌35高效菌株小试生产发酵影响因子的研究", 《西北农业学报》 * |
| 窦霁虹、李博海、孙珺: "苏云金芽孢杆菌发酵实验参数的分析", 《生物数学学报》 * |
| 黄芳一等: "《发酵工程》", 31 May 2019, 华中师范大学出版 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114591851A (en) * | 2021-12-31 | 2022-06-07 | 广东省科学院化工研究所 | Agricultural microbial agent and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112501083B (en) | 2023-01-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102786333B (en) | Phellinus igniarius bag cultivation medium and method for cultivating phellinus igniarius sporophore by same | |
| CN102550284B (en) | Edible mushroom culturing method | |
| KR101479209B1 (en) | Manufacturing Process of Lentinus edodes Culture | |
| CN108277177B (en) | Streptomyces microflavus solid fermentation medium, preparation method and fermentation method thereof, fermentation product, biocontrol product and application | |
| CN110343017A (en) | A kind of polyglutamic acid gel micro-ball microbial inoculum and its application | |
| KR101828217B1 (en) | The method of culturing and growing Cordyceps sinensis in medium that consist of hemp seed and mineral water | |
| CN105948831B (en) | A method of biological and ecological methods to prevent plant disease, pests, and erosion fertilizer is produced using brewex's grains | |
| CN107586725B (en) | Cordyceps liquid culture medium and method for culturing cordyceps by using same | |
| CN106318875A (en) | Cordyceps sobolifera two-way artificial culture method | |
| CN107164241B (en) | Beauveria bassiana solid culture medium and preparation method thereof | |
| CN112501083B (en) | Bacillus thuringiensis liquid fermentation culture medium and fermentation culture method | |
| CN103910545A (en) | Method for using pig manure waste to produce trichoderma harzianum fertilizer | |
| CN109355197A (en) | For promoting the Promoting bacteria and its microbial organic fertilizer of salt-soda soil alfalfa growing | |
| CN111149619B (en) | Cultivation method of pholiota nameko | |
| CN102960369B (en) | Preparation method of granular paecilomyces lilacinus biological nematocide | |
| CN108029445A (en) | A kind of culture medium and its cultural method using coconut husk mushroom culture | |
| CN106190898A (en) | A kind of industrialization liquid fermentation method of the bacillus cereus JZBC1 dissolving pond dinoflagellate | |
| CN115747082A (en) | Beauveria bassiana solid culture medium and application thereof | |
| CN106350457B (en) | A kind of preparation method of the high cryptogam of muscardine | |
| CN115053916A (en) | Microbial agent capable of improving stability of beauveria bassiana and preparation method thereof | |
| CN115747130A (en) | Culture medium for promoting spore production of metarhizium limacinum Mr006, preparation and application thereof | |
| CN109956837A (en) | A kind of how anti-compound fertilizer of tea seed cake and its preparation method and application | |
| CN114258750A (en) | Method for improving strawberry continuous cropping obstacle soil | |
| CN106497801A (en) | A kind of method that utilization Rhodopseudomonas palustris improves soil microbial community | |
| CN112314330A (en) | Method for culturing Roberts' cordyceps sinensis |
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 |