CN112795490A - Beauveria bassiana solid culture medium and culture method thereof - Google Patents
Beauveria bassiana solid culture medium and culture method thereof Download PDFInfo
- Publication number
- CN112795490A CN112795490A CN202110103448.6A CN202110103448A CN112795490A CN 112795490 A CN112795490 A CN 112795490A CN 202110103448 A CN202110103448 A CN 202110103448A CN 112795490 A CN112795490 A CN 112795490A
- Authority
- CN
- China
- Prior art keywords
- beauveria bassiana
- culture
- parts
- culture medium
- solid culture
- 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.)
- Pending
Links
- 239000007787 solid Substances 0.000 title claims abstract description 95
- 241000751139 Beauveria bassiana Species 0.000 title claims abstract description 94
- 239000001963 growth medium Substances 0.000 title claims abstract description 63
- 238000012136 culture method Methods 0.000 title claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 42
- 235000013312 flour Nutrition 0.000 claims abstract description 38
- 240000008042 Zea mays Species 0.000 claims abstract description 36
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 36
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 36
- 235000005822 corn Nutrition 0.000 claims abstract description 36
- 235000015099 wheat brans Nutrition 0.000 claims abstract description 32
- 239000002994 raw material Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 14
- 238000012258 culturing Methods 0.000 claims abstract description 9
- 239000002023 wood Substances 0.000 claims description 39
- 230000001954 sterilising effect Effects 0.000 claims description 14
- 238000009630 liquid culture Methods 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 11
- 238000004659 sterilization and disinfection Methods 0.000 claims description 11
- 239000008280 blood Substances 0.000 claims description 10
- 210000004369 blood Anatomy 0.000 claims description 10
- 239000004744 fabric Substances 0.000 claims description 10
- 238000012360 testing method Methods 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 8
- 244000061458 Solanum melongena Species 0.000 claims description 7
- 235000002597 Solanum melongena Nutrition 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 238000003892 spreading Methods 0.000 claims description 3
- 230000007480 spreading Effects 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 241000012249 Dendrolimus spectabilis Species 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 12
- 238000000855 fermentation Methods 0.000 description 11
- 230000004151 fermentation Effects 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 241000209094 Oryza Species 0.000 description 9
- 235000007164 Oryza sativa Nutrition 0.000 description 9
- 235000009566 rice Nutrition 0.000 description 9
- 239000000463 material Substances 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 7
- 239000010903 husk Substances 0.000 description 7
- 239000000428 dust Substances 0.000 description 6
- 238000011049 filling Methods 0.000 description 6
- 239000000306 component Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- 241000607479 Yersinia pestis Species 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000028070 sporulation Effects 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 244000228451 Stevia rebaudiana Species 0.000 description 3
- 235000006092 Stevia rebaudiana Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 241001631712 Dendrolimus pini Species 0.000 description 2
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 2
- 235000011613 Pinus brutia Nutrition 0.000 description 2
- 241000018646 Pinus brutia Species 0.000 description 2
- 241001122767 Theaceae Species 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 241000255789 Bombyx mori Species 0.000 description 1
- 241000526900 Camellia oleifera Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 206010061217 Infestation Diseases 0.000 description 1
- 241000981121 Leguminivora glycinivorella Species 0.000 description 1
- 241000346285 Ostrinia furnacalis Species 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 230000000967 entomopathogenic effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
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
- C12N1/14—Fungi; 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
- C12N3/00—Spore forming or isolating processes
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a beauveria bassiana solid culture medium which is prepared from the following raw materials in parts by mass: 5-8 parts of corn flour, 5-30 parts of wheat bran, 5-30 parts of sawdust and 30-35 parts of water; the invention also discloses a solid culture method of the beauveria bassiana, raw materials of the solid culture medium are easily available in source and low in price, and the raw materials can be locally obtained in most areas, so that the production cost is greatly reduced; the method for culturing the beauveria bassiana is simple to operate, low in requirement on production conditions, stable in product quality and high in spore yield, and provides technical support for mass culture and production of the beauveria bassiana.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a beauveria bassiana solid culture medium and a culture method thereof.
Background
Beauveria bassiana is a kind of entomopathogenic fungi with wide range and strong pathogenicity to pest hosts, and is one of the most widely used biological pesticides researched at home and abroad at present due to the characteristics of no harm to natural enemies, environmental friendliness, easiness for parasitizing pests for many years under proper conditions and the like, and has been successfully used for large-scale control of a plurality of important agricultural and forestry pests such as pine moth, corn borer, grub, soybean pod borer and the like, and research shows that 1 multiplied by 10 is adopted for every square meter of soil in fields10Spore treatment, the fungi can survive in the cadavers and soil, causing infestation of the next generation of pests, thus maintaining their persistence in the field.
The mass culture production of the beauveria bassiana is a precondition for meeting the field application, and at present, the mass culture of the beauveria bassiana mainly adopts liquid fermentation, solid fermentation and liquid-solid two-phase fermentation. The fermentation time of the beauveria bassiana liquid is short, the main products are blastospores and mycelia, but the survival time of the two products is short; the solid fermentation and the liquid-solid two-phase fermentation have long time, and can generate conidia with longer storage period. In the existing research and production processes, the solid culture medium used for solid fermentation and liquid-solid two-phase fermentation usually adopts fillers such as corn flour, soybean flour, rice flour, silkworm chrysalis meal, vermiculite, wheat bran, rice hulls and the like, and is mostly mixed by adopting two or more materials. In the prior art, the fermentation period for culturing conidia of beauveria bassiana is long, and the spore content is unstable.
Based on the above, a culture medium and a culture method which are beneficial to mass production of conidia of beauveria bassiana, easy to operate, simple and economical in raw materials and less in mixed bacteria pollution need to be researched and designed.
Disclosure of Invention
The invention aims to provide a beauveria bassiana solid culture medium which takes corn flour, wheat bran and wood dust as substrates, has low price and sufficient sources, and greatly reduces the production cost.
The invention also aims to provide a solid culture method of the beauveria bassiana, which has the advantages of simple operation, short fermentation period, high spore yield and less pollution by other bacteria.
The first of the above objects of the present invention can be achieved by the following technical solutions: the beauveria bassiana solid culture medium is prepared from the following raw materials in parts by mass: 5-8 parts of corn flour, 5-30 parts of wheat bran, 5-30 parts of sawdust and 30-35 parts of water.
Preferably, the mass parts of the raw materials are as follows: 5-7 parts of corn flour, 7-17 parts of wheat bran, 18-28 parts of sawdust and 31-33 parts of water.
Preferably, the weight parts of the raw materials are as follows: corn flour 5, wheat bran 17, wood chips 18 and water 32.
Preferably, the particle size of the wood chips is 5-10 mm, and the wood chips can be various wood chips, preferably pine wood chips.
Preferably, one of the preparation methods of the beauveria bassiana solid culture medium is as follows: weighing corn flour, wheat bran, sawdust and water serving as solid culture media according to the mass ratio, uniformly stirring, filling the mixture into cloth bags, wherein 1 cloth bag is filled with about 2.5kg of mixture, putting all the filled cloth bags into a sterilization cabinet, adding a certain amount of water into the sterilization cabinet in the sterilization process to ensure that the sterilization cabinet can reach saturated water vapor, sterilizing the 2.5kg mixed bag filling materials for about 2.87kg at the temperature of 121-126 ℃ and 0.14MPa for 40-60 min, and cooling for later use.
The second object of the present invention can be achieved by the following technical solutions: a solid culture method of beauveria bassiana comprises the following steps:
(1) preparing a beauveria bassiana seed solution: selecting a beauveria bassiana test tube strain, inoculating the strain into a liquid culture medium after slant culture, and carrying out shaking table shake culture to obtain a beauveria bassiana seed solution;
(2) solid culture of beauveria bassiana: inoculating the beauveria bassiana seed liquid in the step (1) to the solid culture medium, uniformly mixing, spreading uniformly in a gauze shallow-bottom wooden frame to enable the thickness of the solid culture medium to be 5-6 cm, then stacking a plurality of gauze shallow-bottom wooden frames, sealing by plastic paper, maintaining sealed culture at 20-26 ℃ for 2-4 days, removing the plastic paper after mycelia grow fully, performing open culture, and culturing for 5-7 days to obtain a beauveria bassiana solid culture.
In the solid culture method of beauveria bassiana:
preferably, the beauveria bassiana test tube strains in the step (1) are separated and purified from beauveria bassiana test tube strains of pine moth, a culture medium adopted in slant culture is 600mL beef extract peptone agar eggplant bottle culture medium, the culture condition is that the culture is carried out for 7-10 days under the condition of 20-25 ℃ until conidia of the beauveria bassiana grow over the slant of the whole eggplant bottle culture medium for later use.
Preferably, the liquid culture medium in the step (1) is prepared from the following raw materials in parts by mass: 28-30 parts of corn flour, 10-12 parts of white sugar and 600-620 parts of water, cooling the corn flour, the white sugar and the water for inoculation after being treated by a high-pressure steam sterilization method, placing the inoculated liquid culture medium at 25 ℃ for 150r/min, and performing shake culture on a shaking table for 3-4 days until the liquid culture medium becomes viscous and white sticky substances appear on the wall of a culture container, thereby obtaining the beauveria bassiana seed liquid.
Preferably, one well-grown 600mL eggplant bottle of beauveria bassiana in step (1) can be inoculated with 8-10 bottles (about 600-620 mL in volume) of liquid culture medium.
Preferably, the solid culture medium in the step (2) is firstly put into a cloth bag, then sterilized at 121-126 ℃ and 0.14MPa for 40-60 min, cooled, and inoculated with beauveria bassiana seed liquid, wherein the dosage relationship between the beauveria bassiana seed liquid and the solid culture medium is 100mL: 450 to 950 g.
In production, 1 bottle of 600mL of seed solution can be mixed with 1 bag of 2.8kg of solid medium in a ratio of about 100mL to 466g, and in the case where the culture conditions are stable, 1 bottle of 600mL of seed solution can be mixed with 2 bags of 2.8kg of solid medium in a ratio of about 100mL to 932g, so that the amount of beauveria bassiana seed solution to the solid medium is 100mL: preferably 450 to 950 g.
Preferably, in the step (2), the plastic paper is removed to reduce the culture humidity from more than 90% to 70-75%, and then open culture is carried out to obtain the white caked beauveria bassiana solid culture.
The humidity is reduced from about 90% to 70-75%, because after the beauveria bassiana mycelia are cultured under the high-humidity condition of 90% or above, if the high-humidity condition is kept, other mixed bacteria are easy to grow, the humidity is properly reduced to 70-75%, the mixed bacteria are less, and the mycelia generated by the beauveria bassiana mycelia enter a conidium growth stage.
Preferably, the step (2) mainly adopts gauze shallow wood frame cultivation, which is a gauze bottom shallow frame, the inner dimension of which is 80-100 cm, the width of which is 40-50 cm and the thickness (height) of which is 6-8 cm, can be adjusted according to actual conditions, and the paving thickness is mainly kept to be 5-7 cm.
Further, drying the beauveria bassiana solid culture in the step (2), and directly crushing the beauveria bassiana solid culture to obtain a primary product when the water content of the beauveria bassiana solid culture is lower than 5%, or separating by a spore separator to obtain conidium powder to obtain a beauveria bassiana product. The spore content in the product is calculated by adopting a blood counting plate method.
The solid culture method of the beauveria bassiana strain provided by the invention is simple to operate, high-yield beauveria bassiana conidia can be obtained in a short fermentation time, and the product quality is stable, so that the solid culture method has obvious economic value and good application prospect.
The method for measuring the spore content of the product obtained by the culture method comprises the following steps:
randomly weighing 1g of beauveria bassiana solid culture primary product, putting into a 100mL triangular flask containing 20mL sterile water, adding 50 μ L of 0.05% Tween-80, shaking with vibrator, sampling 1mL with micropipette, and diluting to 10-7And (4) performing a back microscopic examination, calculating the spore content of the test product by using a blood counting plate method, and repeating the steps for three times to obtain an average value.
Compared with the prior art, the invention has the following advantages:
(1) the wood chip particles are used as the solid culture medium substrate, so that the air permeability is increased, the wood chip has a good moisture-keeping effect, the mycelium grows in large amount and rapidly in the pores of the wood chip particles, finally, the beauveria bassiana can grow freely to the inside and outside of the solid culture medium, manual material turning is not needed in the whole culture process, and the time and the labor are saved;
(2) the invention has simple operation process, easy realization of large-scale batch production, low requirement on production conditions and easy control of production quality;
(3) the raw materials of the solid culture medium, namely corn flour, wheat bran and wood dust, are low-price materials, the sources are easy to obtain, local materials can be obtained in southern areas, the transportation cost is reduced, and the culture cost of the beauveria bassiana is greatly reduced;
(4) the method adopts the gauze shallow-bottom wood frame stacked culture, has obvious advantages, the sealed culture is required to be kept at the initial stage of the beauveria bassiana solid culture, the wood frames are stacked on the frames, the frames are surrounded and covered by the plastic paper, the sealed culture can be kept, the humidity is required to be reduced after the mycelia grow fully, the plastic paper is only required to be removed, the formation and the mature shedding of conidia are facilitated after the humidity is reduced, the closed culture can be quickly changed into the open culture by adopting the gauze shallow-bottom wood frame stacked culture, the beauveria bassiana can form a white caked beauveria bassiana solid culture in a relatively short time, and the pollution of infectious microbes is reduced.
Drawings
The invention is further illustrated by the following figures.
Fig. 1 is a low-bottom wooden frame of gauze used in the present application.
Detailed Description
The present invention is further illustrated by the following specific examples.
The effect of different components of the solid medium on the spore production of beauveria bassiana is first illustrated by example 1 and comparative examples 1-3.
At present, corn flour, wheat bran, rice husks and water are used as raw materials for production, wherein the rice husks are used as fillers and mainly have the functions of loosening and ventilating and providing mycelium attachment, the effect of culturing beauveria bassiana by replacing the rice husks with different filling materials is tested, the test finds that the effect of culturing the beauveria bassiana by replacing the rice husks with wood chips is not wrong, the culture effect of the beauveria bassiana with different wood chip addition amounts is further tested, and the contrast of replacing the rice husks with the wood chips is realized.
Example 1
The beauveria bassiana solid culture medium provided by the embodiment is prepared from the following raw materials in parts by mass: corn flour 5, wheat bran 28, wood chips 7 and water 32.
The particle size of the wood chips is 8mm mesh.
The wood chips are pine wood chips.
The preparation method of the solid culture medium comprises the following steps: weighing corn flour, wheat bran, sawdust and water as solid culture medium components in parts by mass, uniformly stirring to obtain a mixture, filling the mixture into cloth bags, filling 2.5kg of the mixture into 1 cloth bag, putting all the filled cloth bags into a sterilization cabinet, adding a certain amount of water into the sterilization cabinet in the sterilization process to ensure that the sterilization cabinet can reach saturated water vapor, sterilizing at 126 ℃ and 0.14MPa for 60min, and cooling for later use (the weight of 1 cloth bag is 2.8kg after cooling).
The solid culture method for beauveria bassiana by adopting the solid culture medium comprises the following steps:
(1) preparation of beauveria bassiana seed liquid
Inoculating a beauveria bassiana test tube strain separated and purified from pine caterpillars (obtained by adopting a conventional separation and purification step) to a 60mL beef extract peptone agar eggplant bottle culture medium, and culturing for 7-10 days at 25 ℃ until conidia of the beauveria bassiana grow to cover the inclined plane of the whole eggplant bottle culture medium for later use;
filling a liquid culture medium with 600mL of water, 30g of corn flour and 10g of white sugar into a conical flask with the volume of 1000mL, carrying out high-pressure steam sterilization treatment, cooling for inoculation, inoculating 8-10 bottles of liquid culture medium into a well-grown beauveria bassiana bottle, placing the inoculated liquid culture medium at 25 ℃ for 150r/min, and carrying out shake culture on a shaking table for 3-4d until the liquid culture medium becomes viscous and white sticky substances appear on the wall of the conical flask, thus obtaining the beauveria bassiana seed liquid;
(2) solid culture of beauveria bassiana
Inoculating 600mL of seed solution into 1 cloth bag solid material (2.8kg), uniformly mixing, pouring into 1 gauze net shallow bottom wood frame (the gauze net shallow bottom wood frame is shown in figure 1), uniformly spreading to ensure that the thickness of the culture base material is 5-6 cm, stacking the inoculated solid culture frames on a rack, covering the rack by plastic paper, maintaining closed culture at 20-26 ℃, culturing for 2-4 days, removing the plastic paper after the mycelium grows fully, adopting open culture to reduce the culture humidity, and culturing for 5-7 days to obtain a white caked white muscardine fungus solid culture;
(3) beauveria bassiana product collection treatment
And (3) airing the beauveria bassiana solid culture in a dry and ventilated place, and crushing the beauveria bassiana solid culture to obtain an initial product when the water content of the culture material is lower than 5%.
And (3) measuring and calculating the spore content by adopting a blood counting plate method: randomly weighing 1g of a beauveria bassiana solid culture initial product, putting the beauveria bassiana solid culture initial product into a 100mL triangular flask filled with 20mL sterile water, adding 50 mu L of 0.05% Tween-80, shaking the mixture evenly by a vibrator, sampling 1mL by a micropipettor, performing microscopic examination after diluting the mixture to 10-7 in a gradient manner, and calculating the spore content of the test product by a blood count plate method. The average was taken in triplicate.
And (3) measuring results: counting 3 times by using a blood counting plate and taking an average value, wherein the conidium content of the culture is 137.3 multiplied by 108Per gram.
Comparative example 1
In contrast to example 1, the solid medium was: corn flour in parts by mass: wheat bran: stevia rebaudiana residue: water 5: 28: 7: 32 preparing 3 parts of solid culture medium, wherein each part of solid culture medium comprises 360g of corn flour 25g, wheat bran 140g, stevia rebaudiana residue 35g and water 160g, the temperature is 121.3 ℃, the pressure is 103.4kPa, the time is 60min, and the solid culture medium is cooled for standby.
(5) And (3) measuring results: counting 3 times by using a blood counting plate and taking an average value, wherein the conidium content of the culture is 94.4 multiplied by 108Per gram.
Comparative example 2
In contrast to example 1, the solid medium was: corn flour in parts by mass: wheat bran: oil tea shell: water 5: 28: 7: 32 preparing 3 parts of solid culture medium, wherein each part of solid culture medium comprises 360g (25 g of corn flour, 140g of wheat bran, 35g of camellia oleifera shell and 160g of water) and is cooled for standby after being cooled at the temperature of 121.3 ℃ and the pressure of 103.4kPa for 60 min.
(5) The measurement result is that 3 times of counting of a blood counting plate is averaged, and the conidium content of the culture is 62.6 multiplied by 108Per gram.
Comparative example 3
In contrast to example 1, the solid medium was: corn flour in parts by mass: wheat bran: rice husks: water 5: 28: 7: 32 preparing 3 parts of solid culture medium, wherein each part of solid culture medium comprises 360g (25 g of corn flour, 140g of wheat bran, 35g of chaff and 160g of water) at 121.3 ℃ and 103.4kPa for 60min, and cooling for later use.
(5) The measurement result is that 3 times of counting of a blood counting plate is averaged, and the conidium content of the culture is 108.4 multiplied by 108Per gram.
As shown in Table 1, the spore yields of beauveria bassiana on solid media containing wood chips were greatly different from each other, and the influence of the mixture ratio of the components of the solid media containing wood chips on the spore yield was further investigated by the following examples 2 to 3.
TABLE 1 sporulation yield of Beauveria bassiana on different solid media
| Composition of culture medium | The proportion (mass ratio) of the components | Sporulation yield (10)8A/g) |
| Example 1: corn flour, wheat bran, wood dust and water | 5:28:7:32 | 137.3 |
| Comparative example 1: corn flour, wheat bran, stevia rebaudiana residue and water | 5:28:7:32 | 94.4 |
| Comparative example 2: corn flour, wheat bran, oil tea shell and water | 5:28:7:32 | 62.6 |
| Comparative example 3: corn flour, wheat bran, rice husk and water | 5:28:7:32 | 108.4 |
Example 2
In contrast to example 1, the solid medium was: corn flour in parts by mass: wheat bran: wood chip: water 5: 7: 28: 32 preparing 3 parts of solid culture medium, wherein each part of solid culture medium comprises 360g (25 g of corn flour, 35g of wheat bran, 140g of wood chips and 160g of water) and is cooled for standby after being cooled at the temperature of 121.3 ℃ and under the pressure of 103.4kPa for 60 min.
(5) The measurement result is that 3 times of counting of the blood counting plate is averaged, and the conidium content of the culture is 172.6 multiplied by 108Per gram.
Example 3
In contrast to example 1, the solid medium was: corn flour in parts by mass: wheat bran: wood chip: water 5: 17: 18: 32 preparing 3 parts of solid culture medium, wherein each part of solid culture medium comprises 360g (25 g of corn flour, 85g of wheat bran, 90g of wood chips and 160g of water) and is cooled for standby after being cooled at the temperature of 121.3 ℃ and under the pressure of 103.4kPa for 60 min.
(5) The measurement result is that 3 times of counting of a blood counting plate is averaged, and the conidium content of the culture is 201.3 multiplied by 108Per gram.
As shown in Table 2, it can be shown by examples 1 to 3 that, in the solid medium containing wood chips, when the ratio of wood chips to wheat bran is about 1: when 1 hour, the yield of white muscardine fungus is the highest.
TABLE 2 sporulation amounts of the solid Medium Components in different proportions
| Composition of culture medium | The proportion (mass ratio) of the components | Sporulation yield (10)8A/g) |
| Example 1: corn flour, wheat bran, wood dust and water | 5：28：7：32 | 137.3 |
| Example 2: corn flour, wheat bran, wood dust and water | 5：7：28：32 | 172.6 |
| Example 3: corn flour, wheat bran, wood dust and water | 5：17：18：32 | 201.3 |
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The beauveria bassiana solid culture medium is characterized by being prepared from the following raw materials in parts by mass: 5-8 parts of corn flour, 5-30 parts of wheat bran, 5-30 parts of sawdust and 30-35 parts of water.
2. The beauveria bassiana solid culture medium of claim 1, which is characterized in that the mass parts of the raw materials are as follows: 5-7 parts of corn flour, 7-17 parts of wheat bran, 18-28 parts of sawdust and 31-33 parts of water.
3. The beauveria bassiana solid culture medium of claim 2, which is characterized in that the mass parts of the raw materials are as follows: corn flour 5, wheat bran 17, wood chips 18 and water 32.
4. The beauveria bassiana solid medium according to any one of claims 1 to 3, wherein: the particle size of the sawdust is 5-10 mm meshes.
5. A solid culture method of beauveria bassiana is characterized by comprising the following steps:
(1) preparing a beauveria bassiana seed solution: selecting a beauveria bassiana test tube strain, inoculating the strain into a liquid culture medium after slant culture, and carrying out shaking table shake culture to obtain a beauveria bassiana seed solution;
(2) solid culture of beauveria bassiana: inoculating the beauveria bassiana seed liquid in the step (1) to the solid culture medium of any one of claims 1 to 3, uniformly spreading the seed liquid in a gauze shallow wood frame to enable the thickness of the solid culture medium to be 5-6 cm, then stacking a plurality of gauze shallow wood frames, sealing the gauze shallow wood frames by using plastic paper, maintaining closed culture at 20-26 ℃ for 2-4 days, removing the plastic paper after mycelia grow full, performing open culture, and culturing for 5-7 days to obtain a beauveria bassiana solid culture.
6. The solid culture method of beauveria bassiana according to claim 5, characterized in that: the beauveria bassiana test tube strain in the step (1) is separated and purified from beauveria bassiana test tube strain of pine caterpillar, a culture medium adopted in slant culture is 60mL beef extract peptone agar eggplant bottle culture medium, the culture condition is that the culture is carried out for 7-10 days under the condition of 25 ℃, until conidia of the beauveria bassiana grow over the slant of the whole eggplant bottle culture medium for later use.
7. The solid culture method of beauveria bassiana according to claim 5, characterized in that: the liquid culture medium in the step (1) is prepared from the following raw materials in parts by mass: 28-30 parts of corn flour, 10-12 parts of white sugar and 600-620 parts of water, cooling the corn flour, the white sugar and the water for inoculation after being treated by a high-pressure steam sterilization method, placing the inoculated liquid culture medium at 25 ℃ for 150r/min, and performing shake culture on a shaking table for 3-4 days until the liquid culture medium becomes viscous and white sticky substances appear on the wall of a culture container, thereby obtaining the beauveria bassiana seed liquid.
8. The solid culture method of beauveria bassiana according to claim 5, characterized in that: putting the solid culture medium in the step (2) into a cloth bag, sterilizing at 121-126 ℃ and 0.14MPa for 40-60 min, cooling, inoculating a beauveria bassiana seed solution, wherein the dosage relationship between the beauveria bassiana seed solution and the solid culture medium is 100mL: 450 to 950 g.
9. The solid culture method of beauveria bassiana according to claim 5, characterized in that: and (3) removing the plastic paper in the step (2) to reduce the culture humidity from more than 90% to 70-75%, and then carrying out open culture to obtain the white caked beauveria bassiana solid culture.
10. The solid culture method of beauveria bassiana according to claim 5, characterized in that: and (3) drying the beauveria bassiana solid culture in the step (2), and directly crushing the beauveria bassiana solid culture to obtain an initial product when the water content of the beauveria bassiana solid culture is lower than 5%, or separating by using a spore separator to obtain conidium powder to obtain a high-spore beauveria bassiana product, wherein the spore content in the product is calculated by adopting a blood counting chamber method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110103448.6A CN112795490A (en) | 2021-01-26 | 2021-01-26 | Beauveria bassiana solid culture medium and culture method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110103448.6A CN112795490A (en) | 2021-01-26 | 2021-01-26 | Beauveria bassiana solid culture medium and culture method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112795490A true CN112795490A (en) | 2021-05-14 |
Family
ID=75811808
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110103448.6A Pending CN112795490A (en) | 2021-01-26 | 2021-01-26 | Beauveria bassiana solid culture medium and culture method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112795490A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114438011A (en) * | 2022-03-07 | 2022-05-06 | 广西大学 | Fermentation production method and application of beauveria bassiana PfBb spore powder |
| CN118638651A (en) * | 2024-08-15 | 2024-09-13 | 四川躬药科技有限责任公司 | Fermentation process of beauveria bassiana spore powder of medicinal stiff silkworm |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020006650A1 (en) * | 1993-10-12 | 2002-01-17 | Clifford A. Bradley | Solid culture substrate including barley |
| CN106282087A (en) * | 2016-10-28 | 2017-01-04 | 广西壮族自治区林业科学研究院 | A kind of solid medium improving pine moth muscardine sporulation quantity and cultural method |
| CN107299069A (en) * | 2017-08-09 | 2017-10-27 | 郑州大学 | Agricultural microorganism preparation and its application in terms of melon root-knot nematode and watermelon blight is prevented and treated |
| CN111700074A (en) * | 2020-07-08 | 2020-09-25 | 潍坊中创生物科技有限公司 | Preparation method and application of nematophagous fungus chlamydospore wettable powder |
-
2021
- 2021-01-26 CN CN202110103448.6A patent/CN112795490A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020006650A1 (en) * | 1993-10-12 | 2002-01-17 | Clifford A. Bradley | Solid culture substrate including barley |
| CN106282087A (en) * | 2016-10-28 | 2017-01-04 | 广西壮族自治区林业科学研究院 | A kind of solid medium improving pine moth muscardine sporulation quantity and cultural method |
| CN107299069A (en) * | 2017-08-09 | 2017-10-27 | 郑州大学 | Agricultural microorganism preparation and its application in terms of melon root-knot nematode and watermelon blight is prevented and treated |
| CN111700074A (en) * | 2020-07-08 | 2020-09-25 | 潍坊中创生物科技有限公司 | Preparation method and application of nematophagous fungus chlamydospore wettable powder |
Non-Patent Citations (5)
| Title |
|---|
| RAO, P. V.等: ""Selection of substrates for mass multiplication of Beauveria bassiana using semi solid and liquid media"", 《PLANT DISEASE RESEARCH (LUDHIANA)》 * |
| YU, H等: ""Novel strategies for the biocontrol of noctuid pests (Lepidoptera) based on improving ascovirus infectivity using Bacillus thuringiensis"", 《INSECT SCIENCE》 * |
| 季香云: ""白僵菌固体发酵及对蔬菜害虫毒力的研究"", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 张洁等: ""球孢白僵菌固态罐发酵产孢培养的研究"", 《生物学杂志》 * |
| 郭宗芳等: ""白僵菌在油茶苗圃地的宿存变化、介质影响及毒力分析"", 《现代园艺》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114438011A (en) * | 2022-03-07 | 2022-05-06 | 广西大学 | Fermentation production method and application of beauveria bassiana PfBb spore powder |
| CN114438011B (en) * | 2022-03-07 | 2024-03-22 | 广西大学 | Fermentation production method and application of beauveria bassiana PfBb spore powder |
| CN118638651A (en) * | 2024-08-15 | 2024-09-13 | 四川躬药科技有限责任公司 | Fermentation process of beauveria bassiana spore powder of medicinal stiff silkworm |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104025908B (en) | Ganoderma lucidum cultivation method | |
| CN101338279B (en) | Hyphomycetes mite-killing preparation | |
| CN103865810A (en) | Method for culturing golden flower fungus powder by utilizing tea leftovers | |
| CN103404370A (en) | Method for cultivating edible mushroom strains by using lava | |
| CN112795490A (en) | Beauveria bassiana solid culture medium and culture method thereof | |
| CN105316243A (en) | Preparing method and application of composite biological agent for agricultural root-knot nematodes | |
| CN111548944B (en) | Solid fermentation medium for promoting spore production of metarhizium reinhardtii and preparation method and application thereof | |
| KR20140046515A (en) | Manufacturing process of lentinus edodes culture | |
| Zhou | Cultivation of Ganoderma lucidum | |
| CN107164241B (en) | Beauveria bassiana solid culture medium and preparation method thereof | |
| CN109609387A (en) | A kind of fast culture process of Bursaphelenchus xylophilus inner parasitic epiphyte Esteya vermicola | |
| CN104651294B (en) | The fermentation medium and its preparation and application of suitable Metarhizium anisopliae production spore | |
| CN108148765A (en) | One plant of acidproof plan trichodermaharzianum and its application on Rhizoctonia solani Kuhn is inhibited | |
| KR20110006267A (en) | Raw material composite for culture of agaric mushroom and mathod for cultering agaric mushroom using the same | |
| CN100400644C (en) | Bacterial strain and its formulation for preventiong sting type trophi insect | |
| CN113875496A (en) | Rejuvenation method of cordyceps militaris strains | |
| CN111471600B (en) | Culture medium for preparing metarhizium anisopliae spore powder and preparation method of metarhizium anisopliae high spore powder | |
| CN116555050B (en) | Method for propagating Metarhizium anisopliae Mr006 and application thereof | |
| CN112680365A (en) | Liquid culture medium for beauveria bassiana and preparation method of beauveria bassiana microbial inoculum | |
| CN114854665B (en) | Production method and application of paecilomyces lilacinus microsclerotia | |
| CN102972204A (en) | Artificial cultivating method for edible mushrooms | |
| WO2022100701A1 (en) | Method for cultivating ophiocordyceps robertsii | |
| CN102648714A (en) | Method for preparing biopesticide through liquid fermentation | |
| CN115806880A (en) | Beauveria bassiana, separation and screening method thereof and application of beauveria bassiana in controlling bitter orange insect damage | |
| CN110305801B (en) | Method for culturing beauveria bassiana spore powder by using fungus bags |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210514 |
|
| RJ01 | Rejection of invention patent application after publication |