CN107164241A - A kind of muscardine solid medium and preparation method thereof - Google Patents
A kind of muscardine solid medium and preparation method thereof Download PDFInfo
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- CN107164241A CN107164241A CN201710493346.3A CN201710493346A CN107164241A CN 107164241 A CN107164241 A CN 107164241A CN 201710493346 A CN201710493346 A CN 201710493346A CN 107164241 A CN107164241 A CN 107164241A
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Abstract
The invention provides a kind of muscardine solid medium, matrix is used as using corn particle.Present invention also offers the preparation method of muscardine solid medium and the cultural method of muscardine.The present invention is used as muscardine solid culture based substrate using corn particle, raw material sources are easy to get, price is relatively low, most areas can gather materials on the spot, reduce freight, production cost is greatly reduced, is adapted to different scales enterprise and research unit carries out muscardine research and production, with significant economic value and good application prospect.Meanwhile, the solid medium that the present invention is provided can be used for muscardine solid fermentation and liquid-solid two-phase zymotechnique, and cultural method is easy to operate, and fermentation period is short, and sporulation quantity is high.
Description
Technical field
The invention belongs to muscardine culture technique field, more particularly to a kind of muscardine solid medium and its preparation side
Method.
Background technology
Muscardine is that a class is wide to agriculture and forestry injurious insect host range and pathogenic strong insect pathogenic fungus, Yin Qiyi cultures,
The features such as easily epidemic disease not being formed under insect parasite, environmentally friendly, suitable condition, as current most study application both at home and abroad
Most wide insect biocontrol disease fungus, is successfully used for mass control pine moth, corn borer, grub and eating-core bean worm etc. many
Plant important agriculture and forestry insect.
At present, the major way of muscardine mass propgation has liquid fermentation, solid fermentation and liquid-solid two-phase fermentation.Muscardine
The liquid fermentation time is short, and primary product is blastopore and mycelium, but the time-to-live of both products is shorter, should not process
For dosage form；Solid fermentation and liquid-solid two-phase fermentation time are long, but can produce storage period longer conidium.It is existing to grind
Study carefully and in production technology, solid fermentation and the used solid medium of liquid-solid two-phase fermentation generally select bean powder, corn flour, silkworm
Pupa powder, wheat bran, fine rice bran or bagasse and other agricultural byproducts are matrix, many to be mixed using two kinds and above matrix, each component
Ratio need to be controlled strictly, while also need to add vermiculite, the inserts such as rice hulls, to play hydrofuge, ventilation during the fermentation
Effect.
The content of the invention
In view of this, it is an object of the invention to provide a kind of muscardine solid medium and preparation method thereof, the present invention
The muscardine solid medium of offer is cheap, and source is sufficient, and during for muscardine culture, fermentation period is short, sporulation quantity
It is high.
The invention provides a kind of muscardine solid medium, matrix is used as using corn particle.
The present invention is using corn particle as muscardine solid culture based substrate, and raw material sources are easy to get, and price is relatively low, numerously
Area can gather materials on the spot, and reduce freight, greatly reduce production cost, be adapted to different scales enterprise and research unit enters
Row muscardine is studied with producing, with significant economic value and good application prospect.
In the present invention, the corn particle can be the corn particle after crush maize, and its particle diameter is 2mm~4mm, is contained
Water is 20%~30%.
In the present invention, the solid medium also includes nitrogen source；The nitrogen source is selected from peanut powder, bean powder, fish meal, yeast
Powder or beef extract, addition are 0.2~1wt% of corn particle.
In the present invention, the solid medium also includes inorganic salts, and the inorganic salts include KH2PO4、MgSO4·7H2O
And KNO3.The inorganic salts are added in corn particle in form of an aqueous solutions, and inorganic salt solution includes 0.05wt%'s
KH2PO4, 0.01wt% MgSO4·7H2O, 0.2wt% KNO3, added by 0.2%~0.6wt% of corn particle.
Present invention also offers a kind of preparation method of muscardine solid medium, including：
Corn particle is soaked, pulls out after control water oiling stirs and sterilizes, obtain solid medium.
In the present invention, the corn particle is obtained by corn after pulverizer is crushed, and its particle diameter is 2mm~4mm, greatly
Little particle mixes.
Corn particle is soaked in water makes it absorb water, and the temperature of the immersion is normal temperature, is dipped to corn particle aqueous
Measure as 20%~30%, preferably to 25%.Corn particle is pulled out and excessive moisture is drained, adding a small amount of vegetable oil prevents subsequently
Corn particle sticks agglomerating in sterilization process, and the oil can be salad oil, and addition is 0.5% or so of corn particle weight.
Nitrogen source and/or inorganic salt solution can also be added before being sterilized after corn particle is well mixed with oil, mixing is equal
It is even.Wherein, the nitrogen source be selected from peanut powder, bean powder, fish meal, dusty yeast or beef extract, addition for corn particle 0.2~
1wt%.The inorganic salt solution includes 0.05wt% KH2PO4, 0.01wt% MgSO4·7H2O, 0.2wt% KNO3,
Added by 0.2%~0.6wt% of corn particle.
After well mixed, mixed material is loaded and sterilized in container, the temperature of the sterilizing is 121~125 DEG C；Pressure
Power is 0.1~0.15MPa；Time is 30~60 minutes.Sterilizing can obtain muscardine solid medium after finishing.
The muscardine solid medium both can be used for the solid fermentation of muscardine, can be used for the liquid solid two of muscardine
Mutually ferment.
Present invention also offers a kind of cultural method of muscardine, including：
Muscardine is seeded in the solid medium described in above-mentioned technical proposal and carries out fermented and cultured.
Obtain after solid medium, by muscardine inoculation liquid inoculated and cultured.
Wherein, muscardine inoculation liquid can be dense to be accessed in the form of spore suspension, mycelium or blastopore in solid medium
Spend for 5 × 106~5 × 107Individual/mL, inoculum concentration is 10~20% (V/W).
In the present invention, the temperature of the fermented and cultured is 20~26 DEG C, can be using the closed training such as packed or box-packed
Support, also can be using open cultures such as tray, culture ponds.
In one embodiment, during fermented and cultured, culture closed first, after mycelium is covered with, in pallet
Using open culture.Closed culture is used in vegetative stage, it is ensured that the humidity of compost maintains higher level,
While satisfaction the need for mycelial growth, the probability of culture medium pollution can also be reduced；Compost is poured on pallet by the production spore stage
Middle to use open culture, now solid material has been covered with muscardine mycelia, is difficult pollution microbes, while culture humidity can be reduced, has
Come off beneficial to conidial formation and maturation.
After the completion of above-mentioned culture, solid medium is dried in dry ventilation, when Compost moisture content is less than 5%,
Conidial powder can be obtained by a point spore device separation, be processed as the formulations such as wettable powder, pulvis or oil suspending agent；Can also be direct
Compost progress separating twice is applied directly to field, insecticidal action is both played, compost can also be used as soil and fertilizer.
Compared with prior art, the present invention has advantages below and beneficial effect：
(1) present invention is using corn as solid medium, and because corn particle has certain particle diameter in itself, culture medium is in itself
Certain gap is formed, therefore without additionally ventilating in incubation, without adding other fillers increase gas permeability.
(2) the invention operating process is relatively simple, easily realizes that scale is produced in batches, working condition and production process can people
For regulation and control, the quality of production is easily controlled.Solid fermentation matrix can obtain the higher conidial powder of content through point spore device, further
It is processed as a variety of formulations such as wettable powder；Can also directly by the direct separating twice of corn compost, by spreading fertilizer over the fields, ditch spread or
Spread manuer in holes and be administered to field, using more convenient.
(3) solid medium main matrix of the present invention is corn, relative to other solid mediums of muscardine, raw material sources
It is easy to get, price is relatively low, most areas can gather materials on the spot, and reduce freight, greatly reduce production cost.It is adapted to difference
Large-scale enterprises and research unit carry out muscardine research and production, with significant economic value and good application prospect.
(4) solid medium that the present invention is provided can be used for muscardine solid fermentation and liquid-solid two-phase zymotechnique, culture
Method is easy to operate, and fermentation period is short, and sporulation quantity is high.
Embodiment
Embodiment 1 is inoculated with the solid culture method of muscardine spore suspension
It is prepared by 1 spore suspension
1.1 actication of culture：By the muscardine bacterial strain JCF (deposit number is CGMCC No.11338) of Cord blood in test tube
Activation culture on inclined-plane, activation medium used is general SDAY culture mediums, and condition of culture is 26 ± 1 DEG C, and inclined-plane covers with spore
It can be used after son.
It is prepared by 1.2 spore suspensions：Used after conidium in 1.1 on inclined-plane is transferred into SDAY plating mediums, production spore
Spore suspension is made in 0.1% Tween-80 rinsed with sterile water spore, concussion suspension, and adjustment concentration is 1 × 107Spore/mL.
2 solid cultures
2.1 solid culture matrix manufacturings：Corn breaks into broken particle with pulverizer, and size is 2~4mm or so, soaks 4h,
Average moisture content is 25wt%, pulls control out and removes excessive moisture, plus 0.2% fish meal as nitrogen source, plus 0.5% salad oil is with anti-sticking
Even, it is dispensed into after stirring in the polypropylene PP polybags of 40cm × 22cm sizes, per packed 400g wet feeds.With diameter 4cm,
Long 6cm polypropylene PP pipe tyings, the mouth of pipe is sealed with three layers of cotton and sealed membrane successively, 121 DEG C of high-temp steam sterilizing 40min,
Form solid medium.
2.2 inoculations and culture：Sterilized solid medium is placed in transfer room, after cooling in aseptic operating platform
In access muscardine spore suspension in 10% (v/w) ratio and shake off uniform, remove outermost layer sealed membrane, solid material tiled and deployed
It is placed in incubator, is cultivated under the conditions of 24 ± 2 DEG C, increased without extra in humidity, 24h~48h, stirs 2 solid material to increase
Plus throughput is expected in bottom admittedly, and while can also avoid accumulation of heat in fermentation process from " imitation frosted glass " phenomenon occur.During 4~5d, treat solid
Body compost is covered with after muscardine mycelia, and solid culture medium is poured in pallet and carries out open culture, is conducive to spore to come off,
Solid material thickness is advisable with 3~4cm, and now incubator humidity should be less than 50%.
The processing of 2.3 composts：Muscardine is formed after conidium, and solid material is placed in ventilation or drying room and dried, Gu
Material water content can obtain dust base after being crushed when being less than 5%.
2.4 cultivation results：The solid fermentation cycle is 8d, after solid culture medium dries, 3g is weighed at random and is placed on equipped with glass
In the Tween-80 water of 100ml 0.1% of pearl, concussion makes conidium fully elute, and blood counting chamber is counted, and 3 measurements are made even
Average, culture conidium content is 1.8 × 1010Individual/gram material.
Embodiment 2 is inoculated with the mycelial solid culture method of muscardine
It is prepared by 1 mycelium seed liquor：
1.1 actication of culture：Method be the same as Example 1.
It is prepared by 1.2 bacteria suspensions：Method be the same as Example 1, spore suspension concentration is 5 × 107Spore/mL.
1.3 Shaking culture：Load SDY culture mediums 100ml, 121 DEG C of high-temp steam sterilizing 30min in 500ml triangular flasks,
After cooling, in aseptic operating platform, spore suspension, 26 ± 1 DEG C, 200rpm isothermal vibration cultures, culture are accessed by 1% inoculum concentration
After 20h, the seed liquor that mycelium can be used as muscardine solid culture is formed.
2 solid cultures
2.1 solid culture matrix manufacturings：Method be the same as Example 1, nitrogen source is 0.8% peanut powder.
2.2 inoculations and culture：The solid culture medium being well mixed after inoculation is poured in the culture box of sterilization and cultivated,
Other method be the same as Example 1.
The processing of 2.3 composts：Method be the same as Example 1.
2.4 cultivation results：The solid fermentation cycle is 6d, and blood counting chamber is counted, and 3 measurements are averaged, culture point
Raw spore content is 4.5 × 1010Individual/gram material.
Embodiment 3 is inoculated with the solid culture method of muscardine blastopore
It is prepared by 1 blastopore seed liquor：
1.1 actication of culture：Method be the same as Example 1.
It is prepared by 1.2 bacteria suspensions：Method be the same as Example 1, spore suspension concentration is 5 × 106Spore/mL.
1.3 Shaking culture：Load SDY culture mediums 100ml, 121 DEG C of high-temp steam sterilizing 30min in 500ml triangular flasks,
After cooling, in aseptic operating platform, spore suspension, 26 ± 1 DEG C, 200rpm isothermal vibration cultures, culture are accessed by 1% inoculum concentration
After 28h, the seed liquor that a large amount of blastopores can be used as solid phase fermentation is formed.
2 solid cultures
2.1 solid culture matrix manufacturings：Be the same as Example 1, nitrogen source is 0.5% bean powder.
2.2 inoculations and culture：Be the same as Example 1.
The processing of 2.3 composts：Be the same as Example 1.
2.4 cultivation results：The solid fermentation cycle is 8d, and blood counting chamber is counted, and 3 measurements are averaged, culture point
Raw spore content is 3.2 × 1010/ gram material.
Embodiment 4 adds inorganic salts in solid medium
It is prepared by 1 mycelium seed liquor：
1.1 actication of culture：Method be the same as Example 1.
It is prepared by 1.2 bacteria suspensions：Method be the same as Example 1, spore suspension concentration is 5 × 107Spore/mL.
1.3 Shaking culture：Method be the same as Example 2.
2 solid cultures
2.1 solid culture matrix manufacturings：Method be the same as Example 1, nitrogen source is 1% beef extract, and wet feed is pressed in solid medium
(composition is 0.05%KH to 0.4% addition inorganic salt solution of weight2PO4, 0.01%MgSO47H2O, 0.2%KNO3)。
2.2 inoculations and culture：Be the same as Example 1.
The processing of 2.3 composts：Be the same as Example 1.
2.4 cultivation results：The solid fermentation cycle is 6d, and blood count plate technique, 3 measurements are averaged, culture point
Raw spore content is 6.5 × 1010/ gram material.
Comparative example 1
To illustrate further, corn fermentation culture medium technique is simple, fermentation period is short, compares muscardine JCF (preservations
Numbering is CGMCC No.11338) in corn fermentation culture medium and rice medium, maize powder medium, common fungus culture medium
SDAY and PDA fermentation level, as a result referring to table 1, the muscardine that table 1 is provided for comparative example of the present invention is in different solid mediums
On growing state and production spore level.
Growing state and production spore level of the muscardine of table 1 on different solid mediums
In table 1, corn culture medium is solid medium prepared by embodiment 1, and rice medium matrix is low value polished rice,
Maize powder medium composition includes the water of 4% corn flour, 1.5% agar and surplus.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of muscardine solid medium, it is characterised in that matrix is used as using corn particle.
2. muscardine solid medium according to claim 1, it is characterised in that the particle diameter of the corn particle is 2mm
~4mm；
The water content of the corn particle is 20%~30%.
3. muscardine solid medium according to claim 1, it is characterised in that also including nitrogen source；
The nitrogen source is selected from peanut powder, bean powder, fish meal, dusty yeast or beef extract, and addition is 0.2~1wt% of corn particle.
4. the muscardine solid medium according to claims 1 to 3 any one, it is characterised in that also including inorganic salts.
5. muscardine solid medium according to claim 4, it is characterised in that the inorganic salts include KH2PO4、
MgSO4·7H2O and KNO3。
6. a kind of preparation method of muscardine solid medium, including：
Corn particle is soaked, control water is pulled out and adds a small amount of vegetable oil to be sterilized after stirring, obtain solid medium.
7. preparation method according to claim 6, it is characterised in that the corn particle is obtained after crush maize, its
Particle diameter is 2mm~4mm；
The temperature of the immersion is normal temperature, and it is 20%~30% to be dipped to corn particle water content.
8. preparation method according to claim 7, it is characterised in that the temperature of the sterilizing is 121~125 DEG C；Pressure
For 0.1~0.15MPa；Time is 30~60 minutes.
9. the preparation method according to claim 6~8 any one, it is characterised in that be additionally added before sterilizing nitrogen source and/or
Inorganic salt solution stirs.
10. a kind of cultural method of muscardine, it is characterised in that including：
Muscardine is seeded to solid medium or claim 6~9 any one described in Claims 1 to 5 any one
Cultivated in the solid medium that described method is prepared.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111100837A (en) * | 2020-02-12 | 2020-05-05 | 河北省农林科学院植物保护研究所 | Application of ethephon in promotion of spore production of beauveria bassiana |
| CN111548944A (en) * | 2020-06-05 | 2020-08-18 | 福建省农业科学院植物保护研究所 | Solid fermentation medium for promoting spore production of metarhizium reinhardtii and preparation method and application thereof |
| CN114621908A (en) * | 2022-03-16 | 2022-06-14 | 福建省林业科学研究院 | Fermentation method of beauveria bassiana serosa |
| CN115747082A (en) * | 2022-12-23 | 2023-03-07 | 贵州省生物研究所 | Beauveria bassiana solid culture medium and application thereof |
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| CN104115672A (en) * | 2014-07-24 | 2014-10-29 | 巩玉升 | Cultivation method for tomato cordyceps militaris fruits |
| CN105087458A (en) * | 2015-08-11 | 2015-11-25 | 成都易创思生物科技有限公司 | Culture medium and method capable of increasing Beauveria brongniartii sporulation quantity favorably |
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| CN103881930A (en) * | 2014-03-28 | 2014-06-25 | 东北农业大学 | Solid culture medium and culture method of beauveria |
| CN104067922A (en) * | 2014-07-24 | 2014-10-01 | 巩玉升 | Cultivation method for color pepper ganoderma lucidum fruit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111100837A (en) * | 2020-02-12 | 2020-05-05 | 河北省农林科学院植物保护研究所 | Application of ethephon in promotion of spore production of beauveria bassiana |
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| CN111548944B (en) * | 2020-06-05 | 2022-06-14 | 福建省农业科学院植物保护研究所 | Solid fermentation medium for promoting spore production of metarhizium reinhardtii and preparation method and application thereof |
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| CN114621908B (en) * | 2022-03-16 | 2024-03-22 | 福建省林业科学研究院 | Fermentation method of beauveria bassiana serosa |
| CN115747082A (en) * | 2022-12-23 | 2023-03-07 | 贵州省生物研究所 | Beauveria bassiana solid culture medium and application thereof |
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