CN106497805B - A kind of cultural method of beauveria bassiana - Google Patents
A kind of cultural method of beauveria bassiana Download PDFInfo
- Publication number
- CN106497805B CN106497805B CN201611270331.2A CN201611270331A CN106497805B CN 106497805 B CN106497805 B CN 106497805B CN 201611270331 A CN201611270331 A CN 201611270331A CN 106497805 B CN106497805 B CN 106497805B
- Authority
- CN
- China
- Prior art keywords
- vitamin
- culture
- parts
- beauveria bassiana
- cultural method
- 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.)
- Active
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
- C12N1/14—Fungi; Culture media therefor
Abstract
The present invention discloses a kind of cultural method of beauveria bassiana, including (1) primary inclined plane culture: beauveria bassiana thallus is inoculated in primary inclined plane culture medium, 24-25 DEG C culture 5-6 days；(2) secondary liquid culture: the strain obtained after step (1) culture is seeded in seeding tank, in 24-25 DEG C of culture 24-28h, (3) three-level fluid enlargement culture: the strain obtained after step (2) culture is seeded in fermentor, in 25-28 DEG C of culture 28-32h；(4) level Four solid culture: the strain that step (3) expand after culture is inoculated into solid medium, cultivates 5-6d at 22-25 DEG C.By the screening and proportion of nutrient media components in each stage, while cooperating the temperature and humidity condition of culture, cultivation cycle can be shortened, improves the conidial quality of beauveria bassiana and yield, improve conidial activity.
Description
Technical field
The present invention relates to a kind of cultural methods of beauveria bassiana.
Background technique
Muscardine is a kind of ascomycetous entomogenous fungi, and main species include beauveria bassiana and muscardine etc.,
Conidium is often generated by vegetative propagation, mycelia has tabula to have branch.Muscardine can invade 6 Ge Mu, 15 section, more than 200 kinds of elder brothers
Worm, mite class polypide in mass propagation, while generating white stiff plain (Non-ribosomal peptide toxoid), oosporein (benzoquinones poison
Element) and calcium oxalate crystal, these substances insect can be caused to be poisoned, upset metabolism so that dead.Wherein, beauveria bassiana is
Ascomycetous fungi, can industrialize culture production, and people can be processed into microbial insecticide using its conidium generated；
The Chinese medicine larva of a silkworm with batrytis can also be obtained by Infected by Beauveria bassiana silkworm.
The comparison of ingredients of existing beauveria bassiana culture medium is single, bad to the growth result for promoting beauveria bassiana,
The design carried out for the growth course of beauveria bassiana is less, during the cultivation process vulnerable to living contaminants, therefore, obtained ball
The conidial unstable quality of beauveria bassiana, virulence is weaker, and the breeding cycle is longer.
Summary of the invention
In view of this, the application provides a kind of cultural method of beauveria bassiana, pass through nutrient media components in each stage
Screening and proportion, while cooperating the temperature and humidity condition of culture, cultivation cycle can be shortened, it is mitogenetic to improve beauveria bassiana
The quality and yield of spore, improve conidial activity.
In order to solve the above technical problems, technical solution provided by the invention is a kind of cultural method of beauveria bassiana, institute
State cultural method the following steps are included:
(1) primary inclined plane culture: beauveria bassiana thallus is inoculated in primary inclined plane culture medium, is cultivated at 24-25 DEG C
5-6 days；
(2) secondary liquid culture: the strain obtained after step (1) culture is seeded in seeding tank, is cultivated at 24-25 DEG C
The component of 24-28h, the fluid nutrient medium in the seeding tank include: PDA culture medium by weight percentage, supplement 10-20%
Peptone, 5-10% urea, 5-20% sucrose, 10-15% wheat bran, 0.1-0.5% potassium dihydrogen phosphate, 0.01-0.05% sulfuric acid
Magnesium, 0.01-0.05% vitamin B, 0.01-0.05% vitamin C, 0.01-0.02% zinc sulfate, 0.01-0.02% copper sulphate,
0.05-0.1% iron chloride, 0.05-0.1% calcium chloride；
(3) three-level fluid enlargement culture: the strain obtained after step (2) culture is seeded in fermentor, at 25-28 DEG C
28-32h is cultivated, the component of the fluid enlargement culture base in the fermentor includes: PDA culture medium by weight percentage, is mended
Fill 10-20% peptone, 10-20% sucrose, 10-20% soybean powder, 0.1-0.5% potassium dihydrogen phosphate, 0.05-0.1% sulfuric acid
Magnesium, 0.02-0.05% vitamin B, 0.01-0.02% vitamin C, 0.05-0.15% calcium chloride, 0.2-1.0% sodium alginate；
(4) level Four solid culture: the strain that step (3) expand after culture is inoculated into solid medium, at 22-25 DEG C
Lower culture 5-6d, the component of the solid medium include: 20-30 parts of wheat bran, 10-20 parts of dried silkworm chrysalis meal, Huang according to parts by weight
10-20 parts of bean powder, 1-3 parts of vitamin B, 1-2 parts of vitamin C, 2-10 parts of sucrose, 20-40 parts of rice bran；
In step (2) and step (3), the vitamin B is vitamin B1, in vitamin B2, vitamin B3, vitamin B6
At least one；
In step (2) and step (3), the vitamin B is the composition of vitamin B2, vitamin B3, vitamin B6, institute
State vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:(1-2): (1-2).
Preferably, the component of step (1) described slant medium include: according to parts by weight 20-30 parts of potato starch,
1-2 parts of peptone, 5-10 parts of lactose, 1-5 parts of sucrose, 0.05-0.1 parts of potassium dihydrogen phosphate, 0.05-0.1 parts of magnesium sulfate, agar 1-
2 parts.
More preferably, the component of step (1) described slant medium includes: potato starch 25 according to parts by weight
Part, 1 part of peptone, 8 parts of lactose, 3 parts of sucrose, 0.08 part of potassium dihydrogen phosphate, 0.06 part of magnesium sulfate, 2 parts of agar.
Preferably, the component of the fluid nutrient medium in step (2) described seeding tank includes: by weight percentage
PDA culture medium, supplement 18% peptone, 7% urea, 15% sucrose, 13% wheat bran, 0.3% potassium dihydrogen phosphate,
0.02% magnesium sulfate, 0.04% vitamin B, 0.02% vitamin C, 0.015% zinc sulfate, 0.018% copper sulphate, 0.08% chlorine
Change iron, 0.07% calcium chloride.
Preferably, the component of the fluid enlargement culture base in step (3) described fermentor includes: by weight percentage
PDA culture medium, supplement 15% peptone, 18% sucrose, 17% soybean powder, 0.3% potassium dihydrogen phosphate, 0.07% magnesium sulfate,
0.04% vitamin B, 0.03% vitamin C, 0.10% calcium chloride, 0.8% sodium alginate.
More preferably, in step (2) and step (3), the vitamin B is vitamin B2, vitamin B3, vitamin B6
Composition, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:1.5:1.8.
Preferably, step (1) described slant medium first adjusts pH value to 6.5-6.8 before inoculation, then is in pressure
Pressure maintaining sterilizing 40-60min under 100-110kPa.
Preferably, the component of step (4) described solid medium includes: 25 parts of wheat bran, dried silkworm chrysalis meal 15 according to parts by weight
Part, 15 parts of soybean powder, 2 parts of vitamin B, Catergen part, 7 parts of sucrose, 30 parts of rice bran.
Preferably, the humidity of level Four solid culture is 95-98% in step (4).
Preferably, in step (4) solid medium with a thickness of 5-6cm, the ratio that affixed kind of liquid be 1:(1.2-1.3).
Preferably, after covering with conidium in step (4) level Four solid culture to solid medium, place can be dried
Reason.
The PDA culture medium is the abbreviation of potato dextrose agar, i.e. Potato Dextrose Agar
Medium.Its way is: the potato for weighing the clean peeling of 200g is cut into small pieces, and adds boiling is rotten (to boil 20-30min, energy quilt
Glass bar is poked), with eight layers of filtered through gauze, heating, needs further according to experiment plus 1-10g agar, continuation heating stirring are mixed
It is even, after agar has dissolved, glucose is added, stirs evenly, supply moisture again to 1000 milliliters after slightly cooling, dispense test tube or
Person's conical flask is jumped a queue, is wrapped up, and 115 DEG C of sterilizings are taken out test tube pendulum inclined-plane after twenty minutes or shaken up, after cooling to obtain the final product.
In technical scheme, the culture of the beauveria bassiana includes primary inclined plane culture, secondary liquid
The step of culture, three-level fluid enlargement culture, level Four solid culture, by primary inclined plane culture, beauveria bassiana strain is obtained,
The component and proportion of slant medium are controlled, and adds Element Potassium and magnesium needed for growth, while the temperature for controlling culture exists
Between 24-25 DEG C, accelerate the growth of strain；By secondary liquid culture, improve strain quantity rapidly, fluid nutrient medium
Component includes carbon source, nitrogen source necessary to growth, also add potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, copper sulphate,
Iron chloride, calcium chloride provide element needed for thalli growth, while also adding vitamin B and vitamin C, and the two can be improved
Thalli growth speed improves thalline quantity, and the above compounding can greatly improve the speed of growth of strain, improves strain quantity, this
Outside, vitamin B is the compounding of vitamin B2, vitamin B3, vitamin B6, can be improved guarantee bacterial activity, and improve strain
The speed of growth；By three-level fluid enlargement culture, strain continued growth, quantity increases, while strain generates mycelium, liquid
The component that body expands culture medium includes carbon source, nitrogen source necessary to growth, also adds and promotes mycelial growth phosphoric acid
Potassium dihydrogen, magnesium sulfate, calcium chloride, while vitamin B and vitamin C are also added, thallus and mycelium can be improved in the two
The speed of growth, vitamin B are the compounding of vitamin B2, vitamin B3, vitamin B6, can be improved guarantee mycelia active, this
Outside, it also added sodium alginate in the fluid enlargement culture base, can be improved the viscosity of liquid, be conducive to nutrient media components
Suspension, improve culture medium in component utilization rate, while by its match adjustment, make broth viscosity control adapt to model
It encloses, when avoiding liquid sloshing, mycelium shakes generation therewith and pulls, and destroys mycelium；Pass through level Four solid culture, conidium
It is a large amount of to generate, it can be dried at this time, obtain required beauveria bassiana conidia powder, in solid medium, addition
Wheat bran, dried silkworm chrysalis meal, soybean powder, rice bran, sucrose, for mycelium and conidium provide carbon source and nitrogen source, while wheat bran, Huang
The lax quality of bean powder, rice bran has preferable moisture retention and gas permeability, provides wet and ventilative environment, is conducive to mycelia
The quick breeding of body and production spore can accelerate the speed that mycelium produces spore, together in addition, being also added into vitamin B and vitamin C
When with compounding for dried silkworm chrysalis meal conidial activity can be improved.
Compared with prior art, the cultural method of beauveria bassiana described in technical scheme, including primary inclined plane
Culture, secondary liquid culture, three-level fluid enlargement culture, level Four solid culture, pass through the sieve of nutrient media components in each stage
Choosing and proportion, while cooperating the temperature and humidity condition of culture, cultivation cycle can be shortened, improve beauveria bassiana conidium
Quality and yield, improve conidial activity.
Specific embodiment
It is right combined with specific embodiments below in order to make those skilled in the art more fully understand technical solution of the present invention
The present invention is described in further detail.
Embodiment 1
A kind of cultural method of beauveria bassiana described in the present embodiment, the cultural method the following steps are included:
(1) slant medium primary inclined plane culture: is adjusted into pH value to 6.8, then the pressure maintaining sterilizing in the case where pressure is 105kPa
Beauveria bassiana thallus is inoculated in primary inclined plane culture medium by 50min after cooling, is cultivated 5 days at 25 DEG C, the inclined-plane training
The component for supporting base includes: 25 parts of potato starch, 1 part of peptone, 8 parts of lactose, 3 parts of sucrose, biphosphate according to parts by weight
0.08 part of potassium, 0.06 part of magnesium sulfate, 2 parts of agar；
(2) secondary liquid culture: the strain obtained after step (1) culture is seeded in seeding tank, is cultivated at 25 DEG C
The component of 26h, the fluid nutrient medium in the seeding tank include: PDA culture medium by weight percentage, supplement 18% albumen
Peptone, 7% urea, 15% sucrose, 13% wheat bran, 0.3% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.04% vitamin B,
0.02% vitamin C, 0.015% zinc sulfate, 0.018% copper sulphate, 0.08% iron chloride, 0.07% calcium chloride；The dimension life
Plain B is the composition of vitamin B2, vitamin B3, vitamin B6, the weight of the vitamin B2, vitamin B3, vitamin B6
Than for 1:1.5:1.8；
(3) three-level fluid enlargement culture: the strain obtained after step (2) culture is seeded in fermentor, is trained at 28 DEG C
30h is supported, the component of the fluid enlargement culture base in the fermentor includes: PDA culture medium by weight percentage, supplement 15%
Peptone, 18% sucrose, 17% soybean powder, 0.3% potassium dihydrogen phosphate, 0.07% magnesium sulfate, 0.04% vitamin B, 0.03%
Vitamin C, 0.10% calcium chloride, 0.8% sodium alginate；The vitamin B is vitamin B2, vitamin B3, vitamin B6
Composition, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:1.5:1.8；
(4) level Four solid culture: the strain that step (3) expand after culture is inoculated into solid medium, at 24 DEG C
5d is cultivated, the humidity of control level Four solid culture is 96%, and the component of the solid medium includes: wheat bran according to parts by weight
25 parts, 15 parts of dried silkworm chrysalis meal, 15 parts of soybean powder, 2 parts of vitamin B, Catergen part, 7 parts of sucrose, 30 parts of rice bran；Wherein, solid
Culture medium with a thickness of 5.5cm, the ratio that affixed kind of liquid is 1:1.2, is covered in level Four solid culture to solid medium mitogenetic
It after spore, can be dried, after drying process, spore extracting be carried out using cyclonic separation collection spore device, it is white to obtain ball spore
Stiff bacterium conidia powder.
In the present embodiment, the beauveria bassiana conidia powder obtained using above-mentioned cultural method, spore content is 8.8 × 1010/
G, spore germination rate 96%, spore rate living are 98%；The bioassay of spore virulence is carried out with 3 instar larvae of pine moth, after spraying bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, 90% or more humidity) 4 days, there is peak mortality in pine moth, and 7 days
The pine moth death rate is up to 95%.
Embodiment 2
A kind of cultural method of beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
Together.
In the cultural method:
(1) primary inclined plane culture: the component of the slant medium include: according to parts by weight 20 parts of potato starch,
1 part of peptone, 5 parts of lactose, 1 part of sucrose, 0.05 part of potassium dihydrogen phosphate, 0.05 part of magnesium sulfate, 1 part of agar；
(2) secondary liquid culture: the component of the fluid nutrient medium in the seeding tank includes: PDA by weight percentage
Culture medium, supplement 10% peptone, 5% urea, 5% sucrose, 10% wheat bran, 0.1% potassium dihydrogen phosphate, 0.01% magnesium sulfate,
0.01% vitamin B, 0.01% vitamin C, 0.01% zinc sulfate, 0.01% copper sulphate, 0.05% iron chloride, 0.05% chlorination
Calcium；The vitamin B is the composition of vitamin B2, vitamin B3, vitamin B6, the vitamin B2, vitamin B3, dimension life
The weight ratio of plain B6 is 1:1:1；
(3) three-level fluid enlargement culture: the component of the fluid enlargement culture base in the fermentor is by weight percentage
Include: PDA culture medium, supplements 10% peptone, 10% sucrose, 10% soybean powder, 0.1% potassium dihydrogen phosphate, 0.05% sulfuric acid
Magnesium, 0.02% vitamin B, 0.01% vitamin C, 0.05% calcium chloride, 0.2% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, vitamin B6 composition, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:1:1；
(4) level Four solid culture: the component of the solid medium includes: 20-30 parts of wheat bran, silkworm chrysalis according to parts by weight
10 parts of powder, 10 parts of soybean powder, 1 part of vitamin B, 1 part of vitamin C, 2 parts of sucrose, 20 parts of rice bran；Wherein, the thickness of solid medium
Degree is 5.5cm, and the ratio that affixed kind of liquid is 1:1.2.
In the present embodiment, the beauveria bassiana conidia powder obtained using above-mentioned cultural method, spore content is 8.2 × 1010/
G, spore germination rate 95%, spore rate living are 98%；The bioassay of spore virulence is carried out with 3 instar larvae of pine moth, after spraying bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, 90% or more humidity) 6 days, there is peak mortality in pine moth, and 8 days
The pine moth death rate is up to 95%.
Embodiment 3
A kind of cultural method of beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
Together.
In the cultural method:
(1) primary inclined plane culture: the component of the slant medium include: according to parts by weight 30 parts of potato starch,
2 parts of peptone, 10 parts of lactose, 5 parts of sucrose, 0.1 part of potassium dihydrogen phosphate, 0.1 part of magnesium sulfate, 2 parts of agar；
(2) secondary liquid culture: the component of the fluid nutrient medium in the seeding tank includes: PDA by weight percentage
Culture medium supplements 20% peptone, 10% urea, 20% sucrose, 15% wheat bran, 0.5% potassium dihydrogen phosphate, 0.05% sulfuric acid
Magnesium, 0.05% vitamin B, 0.05% vitamin C, 0.02% zinc sulfate, 0.02% copper sulphate, 0.1% iron chloride, 0.1% chlorine
Change calcium；The vitamin B is the composition of vitamin B2, vitamin B3, vitamin B6, the vitamin B2, vitamin B3, dimension
The weight ratio of raw element B6 is 1:2:2；
(3) three-level fluid enlargement culture: the component of the fluid enlargement culture base in the fermentor is by weight percentage
Include: PDA culture medium, supplements 20% peptone, 20% sucrose, 20% soybean powder, 0.5% potassium dihydrogen phosphate, 0.1% sulfuric acid
Magnesium, 0.05% vitamin B, 0.02% vitamin C, 0.15% calcium chloride, 1.0% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, vitamin B6 composition, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:2:2；
(4) level Four solid culture: the component of the solid medium includes: 30 parts of wheat bran, dried silkworm chrysalis meal according to parts by weight
20 parts, 20 parts of soybean powder, vitamin B3 part, Catergen part, 10 parts of sucrose, 40 parts of rice bran；Wherein, the thickness of solid medium
For 5.5cm, the ratio that affixed kind of liquid is 1:1.3.
In the present embodiment, the beauveria bassiana conidia powder obtained using above-mentioned cultural method, spore content is 8.8 × 1010/
G, spore germination rate 97%, spore rate living are 98%；The bioassay of spore virulence is carried out with 3 instar larvae of pine moth, after spraying bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, 90% or more humidity) 4 days, there is peak mortality in pine moth, and 8 days
The pine moth death rate is up to 95%.
Embodiment 4
A kind of cultural method of beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
Together.
In the cultural method:
(1) primary inclined plane culture: the component of the slant medium include: according to parts by weight 20 parts of potato starch,
2 parts of peptone, 5 parts of lactose, 5 parts of sucrose, 0.05 part of potassium dihydrogen phosphate, 0.1 part of magnesium sulfate, 1 part of agar；
(2) secondary liquid culture: the component of the fluid nutrient medium in the seeding tank includes: PDA by weight percentage
Culture medium, supplement 20% peptone, 5% urea, 20% sucrose, 10% wheat bran, 0.5% potassium dihydrogen phosphate, 0.01% magnesium sulfate,
0.05% vitamin B, 0.01% vitamin C, 0.02% zinc sulfate, 0.01% copper sulphate, 0.1% iron chloride, 0.05% chlorination
Calcium；The vitamin B is the composition of vitamin B2, vitamin B3, vitamin B6, the vitamin B2, vitamin B3, dimension life
The weight ratio of plain B6 is 1:1:2；
(3) three-level fluid enlargement culture: the component of the fluid enlargement culture base in the fermentor is by weight percentage
Include: PDA culture medium, supplements 20% peptone, 10% sucrose, 20% soybean powder, 0.1% potassium dihydrogen phosphate, 0.1% sulfuric acid
Magnesium, 0.02% vitamin B, 0.02% vitamin C, 0.05% calcium chloride, 1.0% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, vitamin B6 composition, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:2:1；
(4) level Four solid culture: the component of the solid medium includes: 20 parts of wheat bran, dried silkworm chrysalis meal according to parts by weight
20 parts, 10 parts of soybean powder, 3 parts of vitamin B, 1 part of vitamin C, 10 parts of sucrose, 20 parts of rice bran；Wherein, the thickness of solid medium
Degree is 5.5cm, and the ratio that affixed kind of liquid is 1:1.3.
In the present embodiment, the beauveria bassiana conidia powder obtained using above-mentioned cultural method, spore content is 8.6 × 1010/
G, spore germination rate 95%, spore rate living are 98%；The bioassay of spore virulence is carried out with 3 instar larvae of pine moth, after spraying bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, 90% or more humidity) 5 days, there is peak mortality in pine moth, and 7 days
The pine moth death rate is up to 95%.
Embodiment 5
A kind of cultural method of beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
Together.
In the cultural method:
(1) primary inclined plane culture: the component of the slant medium include: according to parts by weight 30 parts of potato starch,
1 part of peptone, 10 parts of lactose, 1 part of sucrose, 0.1 part of potassium dihydrogen phosphate, 0.05 part of magnesium sulfate, 2 parts of agar；
(2) secondary liquid culture: the component of the fluid nutrient medium in the seeding tank includes: PDA by weight percentage
Culture medium, supplement 10% peptone, 10% urea, 5% sucrose, 15% wheat bran, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate,
0.01% vitamin B, 0.05% vitamin C, 0.01% zinc sulfate, 0.02% copper sulphate, 0.05% iron chloride, 0.1% chlorination
Calcium；The vitamin B is the composition of vitamin B2, vitamin B3, vitamin B6, the vitamin B2, vitamin B3, dimension life
The weight ratio of plain B6 is 1:2:1；
(3) three-level fluid enlargement culture: the component of the fluid enlargement culture base in the fermentor is by weight percentage
Include: PDA culture medium, supplements 10% peptone, 20% sucrose, 10% soybean powder, 0.5% potassium dihydrogen phosphate, 0.05% sulfuric acid
Magnesium, 0.05% vitamin B, 0.01% vitamin C, 0.15% calcium chloride, 0.2% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, vitamin B6 composition, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:1:2；
(4) level Four solid culture: the component of the solid medium includes: 30 parts of wheat bran, dried silkworm chrysalis meal according to parts by weight
10 parts, 20 parts of soybean powder, 1 part of vitamin B, Catergen part, 2 parts of sucrose, 40 parts of rice bran；Wherein, the thickness of solid medium
For 5.5cm, the ratio that affixed kind of liquid is 1:1.2.
In the present embodiment, the beauveria bassiana conidia powder obtained using above-mentioned cultural method, spore content is 8.7 × 1010/
G, spore germination rate 95%, spore rate living are 98%；The bioassay of spore virulence is carried out with 3 instar larvae of pine moth, after spraying bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, 90% or more humidity) 5 days, there is peak mortality in pine moth, and 6 days
The pine moth death rate is up to 95%.
Embodiment 6
A kind of cultural method of beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
Together.
In the cultural method:
(1) slant medium primary inclined plane culture: is adjusted into pH value to 6.5, then the pressure maintaining sterilizing in the case where pressure is 100kPa
Beauveria bassiana thallus is inoculated in primary inclined plane culture medium by 60min after cooling, is cultivated 5 days at 24 DEG C；
(2) secondary liquid culture: the strain obtained after step (1) culture is seeded in seeding tank, is cultivated at 24 DEG C
24h；
(3) strain obtained after step (2) culture is seeded in fermentor, in 25 DEG C of culture 28h；
(4) level Four solid culture: the strain that step (3) expand after culture is inoculated into solid medium, at 22 DEG C
5d is cultivated, the humidity of control level Four solid culture is 95%.
In the present embodiment, the beauveria bassiana conidia powder obtained using above-mentioned cultural method, spore content is 8.6 × 1010/
G, spore germination rate 95%, spore rate living are 98%；The bioassay of spore virulence is carried out with 3 instar larvae of pine moth, after spraying bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, 90% or more humidity) 5 days, there is peak mortality in pine moth, and 7 days
The pine moth death rate is up to 95%.
Embodiment 7
A kind of cultural method of beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
Together.
In the cultural method:
(1) slant medium primary inclined plane culture: is adjusted into pH value to 6.8, then the pressure maintaining sterilizing in the case where pressure is 110kPa
Beauveria bassiana thallus is inoculated in primary inclined plane culture medium by 40min after cooling, is cultivated 5-6 days at 25 DEG C；
(2) secondary liquid culture: the strain obtained after step (1) culture is seeded in seeding tank, is cultivated at 25 DEG C
28h；
(3) strain obtained after step (2) culture is seeded in fermentor, in 28 DEG C of culture 32h；
(4) level Four solid culture: the strain that step (3) expand after culture is inoculated into solid medium, at 25 DEG C
6d is cultivated, the humidity of control level Four solid culture is 98%.
In the present embodiment, the beauveria bassiana conidia powder obtained using above-mentioned cultural method, spore content is 8.7 × 1010/
G, spore germination rate 96%, spore rate living are 98%；The bioassay of spore virulence is carried out with 3 instar larvae of pine moth, after spraying bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, 90% or more humidity) 4 days, there is peak mortality in pine moth, and 6 days
The pine moth death rate is up to 95%.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (8)
1. a kind of cultural method of beauveria bassiana, it is characterised in that: the cultural method the following steps are included:
(1) primary inclined plane culture: beauveria bassiana thallus is inoculated in primary inclined plane culture medium, in 24-25 DEG C of culture 5-6
It；
(2) secondary liquid culture: the strain obtained after step (1) culture is seeded in seeding tank, in 24-25 DEG C of culture 24-
The component of 28h, the fluid nutrient medium in the seeding tank include: PDA culture medium by weight percentage, supplement 10-20% egg
White peptone, 5-10% urea, 5-20% sucrose, 10-15% wheat bran, 0.1-0.5% potassium dihydrogen phosphate, 0.01-0.05% magnesium sulfate,
0.01-0.05% vitamin B, 0.01-0.05% vitamin C, 0.01-0.02% zinc sulfate, 0.01-0.02% copper sulphate,
0.05-0.1% iron chloride, 0.05-0.1% calcium chloride；
(3) three-level fluid enlargement culture: the strain obtained after step (2) culture is seeded in fermentor, is cultivated at 25-28 DEG C
The component of 28-32h, the fluid enlargement culture base in the fermentor include: PDA culture medium by weight percentage, supplement 10-
20% peptone, 10-20% sucrose, 10-20% soybean powder, 0.1-0.5% potassium dihydrogen phosphate, 0.05-0.1% magnesium sulfate,
0.02-0.05% vitamin B, 0.01-0.02% vitamin C, 0.05-0.15% calcium chloride, 0.2-1.0% sodium alginate；
(4) level Four solid culture: the strain that step (3) expand after culture is inoculated into solid medium, is trained at 22-25 DEG C
5-6d is supported, the component of the solid medium includes: 20-30 parts of wheat bran, 10-20 parts of dried silkworm chrysalis meal, soybean powder according to parts by weight
10-20 parts, -3 parts of vitamin B1,1-2 parts of vitamin C, 2-10 parts of sucrose, 20-40 parts of rice bran；
In step (2) and step (3), the vitamin B is the composition of vitamin B2, vitamin B3, vitamin B6, the dimension
Raw element B2, vitamin B3, vitamin B6 weight ratio be 1:1-2:1-2.
2. a kind of cultural method of beauveria bassiana according to claim 1, it is characterised in that: step (1) described inclined-plane
The component of culture medium includes: 20-30 parts of potato starch, 1-2 parts of peptone, 5-10 parts of lactose, sucrose 1-5 according to parts by weight
Part, 0.05-0.1 parts of potassium dihydrogen phosphate, 0.05-0.1 parts of magnesium sulfate, 1-2 parts of agar.
3. a kind of cultural method of beauveria bassiana according to claim 2, it is characterised in that: step (1) described inclined-plane
The component of culture medium includes: 25 parts of potato starch, 1 part of peptone, 8 parts of lactose, 3 parts of sucrose, di(2-ethylhexyl)phosphate according to parts by weight
0.08 part of hydrogen potassium, 0.06 part of magnesium sulfate, 2 parts of agar.
4. a kind of cultural method of beauveria bassiana according to claim 1, it is characterised in that: step (2) described seed
The component of fluid nutrient medium in tank includes: PDA culture medium by weight percentage, supplement 18% peptone, 7% urea,
15% sucrose, 13% wheat bran, 0.3% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.04% vitamin B, 0.02% vitamin C,
0.015% zinc sulfate, 0.018% copper sulphate, 0.08% iron chloride, 0.07% calcium chloride.
5. a kind of cultural method of beauveria bassiana according to claim 1, it is characterised in that: step (3) described fermentation
The component of fluid enlargement culture base in tank includes: PDA culture medium by weight percentage, supplements 15% peptone, 18% sugarcane
Sugar, 17% soybean powder, 0.3% potassium dihydrogen phosphate, 0.07% magnesium sulfate, 0.04% vitamin B, 0.03% vitamin C, 0.10%
Calcium chloride, 0.8% sodium alginate.
6. a kind of cultural method of beauveria bassiana according to claim 1, it is characterised in that: step (1) described inclined-plane
Culture medium first adjusts pH value to 6.5-6.8, then the pressure maintaining sterilizing 40-60min in the case where pressure is 100-110kPa before inoculation.
7. a kind of cultural method of beauveria bassiana according to claim 1, it is characterised in that: level Four is solid in step (4)
The humidity of body culture is 95-98%.
8. a kind of cultural method of beauveria bassiana according to claim 1, it is characterised in that: solid is trained in step (4)
Support base with a thickness of 5-6cm, the ratio that affixed kind of liquid be 1:1.2-1.3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611270331.2A CN106497805B (en) | 2016-12-30 | 2016-12-30 | A kind of cultural method of beauveria bassiana |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611270331.2A CN106497805B (en) | 2016-12-30 | 2016-12-30 | A kind of cultural method of beauveria bassiana |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106497805A CN106497805A (en) | 2017-03-15 |
| CN106497805B true CN106497805B (en) | 2019-07-16 |
Family
ID=58333578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611270331.2A Active CN106497805B (en) | 2016-12-30 | 2016-12-30 | A kind of cultural method of beauveria bassiana |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106497805B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906148A (en) * | 2017-04-25 | 2017-06-30 | 四川省农业科学院蚕业研究所 | A kind of culture medium of beauveria bassiana, the cultural method of beauveria bassiana |
| CN108719292B (en) * | 2018-07-07 | 2021-06-08 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Special synergist for beauveria bassiana biological pesticide and preparation method and application thereof |
| CN109628333A (en) * | 2019-02-28 | 2019-04-16 | 广西地源之本肥业有限公司 | A kind of preparation method for the microorganism biological and ecological methods to prevent plant disease, pests, and erosion agent inhibiting insect pest |
| CN110093283B (en) * | 2019-05-15 | 2020-03-17 | 云南星耀生物制品有限公司 | Beauveria bassiana strain and culture method thereof |
| CN110305801B (en) * | 2019-07-31 | 2021-04-09 | 四川省农业科学院经济作物育种栽培研究所 | Method for culturing beauveria bassiana spore powder by using fungus bags |
| CN110352920B (en) * | 2019-08-30 | 2021-09-14 | 江苏海洋大学 | Method for artificially culturing white muscardine silkworms based on beauveria bassiana |
| CN111676142A (en) * | 2020-04-14 | 2020-09-18 | 中国烟草总公司陕西省公司 | Preparation and application method of beauveria bassiana powder |
| CN114058518A (en) * | 2021-11-26 | 2022-02-18 | 盐城市神微微生物菌种科技有限公司 | Culture method of beauveria bassiana |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206587B (en) * | 2011-04-22 | 2013-06-05 | 中国农业科学院植物保护研究所 | Culture method of sclerotium of beauveria bassiana and application thereof |
| CN105176828B (en) * | 2014-07-16 | 2020-04-28 | 新疆农业大学 | Beauveria bassiana XNBb-04 strain and culture method thereof |
-
2016
- 2016-12-30 CN CN201611270331.2A patent/CN106497805B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106497805A (en) | 2017-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106497805B (en) | A kind of cultural method of beauveria bassiana | |
| CN104025908B (en) | Ganoderma lucidum cultivation method | |
| CN107955794B (en) | High-quality preservation method of cordyceps militaris strains | |
| WO2015180519A1 (en) | Method for cultivating high-cordyceps-polysaccharide cordyceps militaris | |
| CN103981105B (en) | The cultural method of a kind of high yield cordycepic acid Cordyceps militaris (L.) Link. | |
| CN104164367B (en) | Dried silkworm cordyceps militaris and culture method thereof | |
| US20140186927A1 (en) | Process for the Production and Utilization of Chlamydospore Rich Slurry Inoculum | |
| CN105766382A (en) | Antrodia cultivating method capable of improving content of triterpene | |
| CN106804285A (en) | Culture method for improving triterpenoid components of antrodia camphorata | |
| CN104328056A (en) | Trichoderma reesei capable of producing cellulase in high yield and application of trichoderma reesei | |
| CN105110961A (en) | Liquid fermentation culture medium for shiitake mushrooms and method for producing shiitake mushrooms through the same | |
| CN105505992A (en) | Method for producing functional food through probiotics compound biological fermentation | |
| CN106797804A (en) | A kind of method that silkworm using abnormal early stage cultivates Cordyceps militaris | |
| CN104541977B (en) | A kind of Chinese fir board culture method of Antrodia camphorata | |
| CN107156638A (en) | A kind of preparation method of Antilipemic monascus powder | |
| CN106318875A (en) | Cordyceps sobolifera two-way artificial culture method | |
| CN106212044A (en) | A kind of Antrodia Camphorata ware formula cultural method | |
| CN106906148A (en) | A kind of culture medium of beauveria bassiana, the cultural method of beauveria bassiana | |
| CN104673740B (en) | Promote the culture medium and preparation method of Nomuraea rileyi production spore | |
| CN100400644C (en) | Bacterial strain and its formulation for preventiong sting type trophi insect | |
| CN106834123A (en) | A kind of muscardine separates expanding propagation method | |
| CN106034738B (en) | A kind of method with silkworm pupa as host artificial culture silkworm chrysalis cicada fungus | |
| WO2022088280A1 (en) | Cultivating method for silkworm pupa-cultured cordyceps militaris | |
| Zeng et al. | Air pressure pulsation solid state fermentation of feruloyl esterase by Aspergillus niger | |
| CN104472208B (en) | Liquid culture method for cordyceps militaris stroma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | 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 |