CN102796672A - Verticillium lecanii solid fermentation medium, preparation method and application - Google Patents
Verticillium lecanii solid fermentation medium, preparation method and application Download PDFInfo
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- CN102796672A CN102796672A CN2012102884686A CN201210288468A CN102796672A CN 102796672 A CN102796672 A CN 102796672A CN 2012102884686 A CN2012102884686 A CN 2012102884686A CN 201210288468 A CN201210288468 A CN 201210288468A CN 102796672 A CN102796672 A CN 102796672A
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- nutritive medium
- corn cob
- substratum
- verticillium lecanii
- solid fermentation
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Abstract
The invention relates to a Verticillium lecanii solid fermentation medium, a preparation method and an application, which belongs to the biology technical field. The preparation method of the medium comprises the following steps: crushing dried corncob into powder by a pulverizer, then blending the corncob powder and a nutrient solution according to mass ratio of 1:2-1:5, wherein the nutrient solution contains glucose, extract yeast, MgSO4, K2HP04, MnSO4, FeSO4 and KCl as the nutrition components, adjusting the pH value of the nutrient solution to 6.0-7.0 by a NaOH solution, and performing high pressure moisture-heat sterilization to obtain the Verticillium lecanii solid fermentation medium. The solid fermentation medium is used for sporulation culture of Verticillium lecanii. Compared with the traditional entomogenous fungi solid fermentation medium, the Verticillium lecanii solid fermentation medium substantially reduces the production cost and environmental pollution at certain degree brought by the corncob treatment.
Description
Technical field
The present invention relates to a kind of substratum, particularly a kind ofly be used for the substratum that the Verticillium lecanii solid fermentation is cultivated.Belong to biological technical field.
Background technology
Be accompanied by the use of chemical pesticide in recent years, 3R (resistance (resistance), increases wildness (resurgence) and residual (residue) again and has become a whole world difficult problem that generally acknowledge, that need to be resolved hurrily.Verticillium lecanii is wide with its regional distribution, host range is wide, to people and animals and natural enemy insect safety, characteristics such as free from environmental pollution, more and more receive people's attention, become one of biocontrol microorganisms that has development potentiality.
Verticillium lecanii (Lecanicillium lecanii); Belonging to Deuteromycotina (Deuteromycotina) hyphomycetes (Hyphomycetes) Moniliales (Moniliales) Moniliaceae (Moniliaceae) Verticillium and belong to (Lecanicillium), is a kind of regional distribution sick fungal pathogens of worm widely.The host range of Verticillium lecanii is extensive, can parasitic Homoptera, the insect of kind such as Hemiptera, Orthoptera, lepidopteran, Coleoptera, Hymenoptera and Thysanoptera, can also parasitize mite class, nematode and some phytopathogens.Biocontrol microorganisms as greenhouse insect such as aleyrodid, aphid, thrips is widely used.1861, Neitner found to parasitize the Verticillium lecanii on the coffee lecanium first in the Ceylon.The middle and later periods eighties 20th century; States such as Britain, Holland, Russia have appearances such as this bacterium commodity preparation Vertalec, Thriptal and Mycotal in succession; In order to trialeurodes vaporariorum, aphid and the thrips insect on control flowers and the greenhouse vegetable, and obtained good control effect.
Summary of the invention
The object of the present invention is to provide a kind of Verticillium lecanii solid fermentation substratum.
The present invention realizes above-mentioned purpose through adopting following technical scheme:
A kind of Verticillium lecanii solid fermentation of the present invention substratum is characterized in that described substratum is is that 1:2～1:5 is mixed and obtains after the sterilization by corn cob powder and nutritive medium by mass ratio;
Contain glucose 22～28gL in the described nutritive medium
-1, yeast extract paste 22～28gL
-1, MgSO
40.05～0.2gL
-1, K
2HPO
40.5～0.9gL
-1, MnSO
40.05-0.075gL
-1, FeSO
40.005-0.015gL
-1, KCl 0.25～0.4gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.0-7.0.
In the present invention, preferred, described substratum is to be that 1:3 is mixed and obtains after sterilizing by corn cob powder and nutritive medium by mass ratio.
In the present invention, preferred, contain glucose 24～26gL in the described nutritive medium
-1, yeast extract paste 24～26gL
-1, MgSO
40.1～0.15gL
-1, K
2HP0
40.6～0.8gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.3～0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5, and is preferred, contains glucose 26gL in the described nutritive medium
-1, yeast extract paste 25gL
-1, MgSO
40.14gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5.
In the present invention, preferred, described corn cob powder is that the corn cob dried and crushed is become particle diameter is to obtain behind the particle of 2-4mm, and preferably the corn cob dried and crushed being become particle diameter is to obtain behind the particle of 3mm.
Corn cob is the excellent core behind the corn rod threshing.Those skilled in the art can gather voluntarily or purchase obtains from market.
Further, the present invention also provides a kind of method for preparing above each described substratum, it is characterized in that may further comprise the steps:
(1) raw materials pretreatment: corn cob is dried and with kibbler its pulverizing obtained the corn cob powder;
(2) proportion of raw materials: corn cob powder and nutritive medium are that 1:2～1:5 is mixed by mass ratio, contain glucose 22～28gL in the described nutritive medium
-1, yeast extract paste 22～28gL
-1, MgSO
40.05～0.2gL
-1, K
2HP0
40.5～0.9gL
-1, MnSO
40.05-0.075gL
-1, FeSO
40.005-0.015gL
-1, KCl 0.25～0.4gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.0-7.0;
(3) sterilization of substratum: the high pressure moist heat sterilization, obtaining with the corn cob is the Verticillium lecanii solid fermentation substratum of raw material and matrix.
In the present invention, preferred, the corn cob powder described in the step (1) is that the corn cob dried and crushed is become particle diameter is to obtain behind the particle of 2-4mm, and preferably the corn cob dried and crushed being become particle diameter is to obtain behind the particle of 3mm.
In the present invention, preferred, be to be that 1:3 is mixed by mass ratio in the step (2) with corn cob powder and nutritive medium.
In the present invention, preferred, contain glucose 24～26gL in the nutritive medium described in the step (2)
-1, yeast extract paste 24～26gL
-1, MgSO
40.1～0.15gL
-1, K
2HP0
40.6～0.8gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.3～0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5, and is preferred, contains glucose 26gL in the described nutritive medium
-1, yeast extract paste 25gL
-1, MgSO
40.14gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5.
In the present invention, preferred, the high pressure moist heat sterilization described in the step (3) is to be 121 ℃ in temperature, and pressure is the 45-60min that sterilizes under the condition of 103.4205kPa.
Further, the present invention also provides the application of described substratum in the Verticillium lecanii solid fermentation is cultivated.
Characteristics of the present invention: the present invention is culture medium and raw material with the corn cob, produces the carbon source content and the C/N ratio of spore according to suitable Verticillium lecanii and adds nutritive substance glucose and yeast extract paste, has replenished Verticillium lecanii simultaneously and has produced the required inorganic salt of spore.(mainly is rice with agricultural-food waste material corn cob as the raw materials for production of the carrier of substratum and raw material and traditional entomogenous fungi substratum; Cereal etc.) compare and greatly reduce production cost, when obtaining economic benefit, reduced environmental pollution to a certain extent.Substratum of the present invention is applicable to the product spore cultivation of Verticillium lecanii.Cultivate Verticillium lecanii with solid medium of the present invention, can make every gram matrix sporulation quantity reach 3.5～7.5 * 10
9Individual.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
The preparation of embodiment 1 Verticillium lecanii solid fermentation substratum
(1) raw materials pretreatment: corn cob is dried and uses kibbler that its pulverizing is obtained the corn cob powder of particle diameter as 3mm;
(2) proportion of raw materials: corn cob powder and nutritive medium are that 1:3 is mixed by mass ratio, contain glucose 25gL in the described nutritive medium
-1, yeast extract paste 25gL
-1, MgSO
40.15gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.3gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5;
(3) sterilization of substratum: in temperature is 121 ℃, and pressure is high pressure moist heat sterilization 45min under the condition of 103.4205kPa, and obtaining with the corn cob is the Verticillium lecanii solid fermentation substratum of raw material and matrix.
The preparation of embodiment 2 Verticillium lecanii solid fermentation substratum
(1) raw materials pretreatment: corn cob is dried and uses kibbler that its pulverizing is obtained the corn cob powder of particle diameter as 4mm;
(2) proportion of raw materials: corn cob powder and nutritive medium are that 1:4 is mixed by mass ratio, contain glucose 25gL in the described nutritive medium
-1, yeast extract paste 25gL
-1, MgSO
40.15gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.3gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5;
(3) sterilization of substratum: in temperature is 121 ℃, and pressure is high pressure moist heat sterilization 60min under the condition of 103.4205kPa, and obtaining with the corn cob is the Verticillium lecanii solid fermentation substratum of raw material and matrix.
The preparation of embodiment 3 Verticillium lecanii solid fermentation substratum
(1) raw materials pretreatment: corn cob is dried and uses kibbler that its pulverizing is obtained the corn cob powder of particle diameter as 3mm;
(2) proportion of raw materials: corn cob powder and nutritive medium are that 1:5 is mixed by mass ratio, contain glucose 24gL in the described nutritive medium
-1, yeast extract paste 26gL
-1, MgSO
40.1gL
-1, K
2HP0
40.8gL
-1, MnSO
40.06gL
-1, FeSO
40.01gL
-1, KCl 0.4gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.0;
(3) sterilization of substratum: in temperature is 121 ℃, and pressure is high pressure moist heat sterilization 45min under the condition of 103.4205kPa, and obtaining with the corn cob is the Verticillium lecanii solid fermentation substratum of raw material and matrix.
The preparation of embodiment 4 Verticillium lecanii solid fermentation substratum
(1) raw materials pretreatment: corn cob is dried and uses kibbler that its pulverizing is obtained the corn cob powder of particle diameter as 2mm;
(2) proportion of raw materials: corn cob powder and nutritive medium are that 1:2 is mixed by mass ratio, contain glucose 28gL in the described nutritive medium
-1, yeast extract paste 24gL
-1, MgSO
40.2gL
-1, K
2HP0
40.5gL
-1, MnSO
40.075gL
-1, FeSO
40.015gL
-1, KCl 0.25gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 7.0;
(3) sterilization of substratum: in temperature is 121 ℃, and pressure is high pressure moist heat sterilization 45min under the condition of 103.4205kPa, and obtaining with the corn cob is the Verticillium lecanii solid fermentation substratum of raw material and matrix.
The preparation of embodiment 5 Verticillium lecanii solid fermentation substratum
(1) raw materials pretreatment: corn cob is dried and uses kibbler that its pulverizing is obtained the corn cob powder of particle diameter as 3mm;
(2) proportion of raw materials: corn cob powder and nutritive medium are that 1:3 is mixed by mass ratio, contain glucose 26gL in the described nutritive medium
-1, yeast extract paste 25gL
-1, MgSO
40.14gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5;
(3) sterilization of substratum: in temperature is 121 ℃, and pressure is high pressure moist heat sterilization 45min under the condition of 103.4205kPa, and obtaining with the corn cob is the Verticillium lecanii solid fermentation substratum of raw material and matrix.
The application of embodiment 6 solid fermentation substratum of the present invention in the Verticillium lecanii solid fermentation is cultivated
1, material
Verticillium lecanii strain V
4Introduce the full Russia of Russia plant protection institute; Bacterial strain is on the books in the article of having delivered; Document is called " Verticillium lecanii biological characteristics and the research pathogenic to Trialeurodes vaporariorum Westwood thereof "; Be published in volume the 6th phase plant protection periodical December the 28th in 2002, existing by the preservation of plant protection institute of Heilungkiang Academy of Agricultural Sciences, provide.
2, method
On the Bechtop of sterilisable chamber, have mycelial verticillium lecanii strain V with inoculating needle picking fritter
4Agar block move on to sterilize and be equipped with in the triangular flask of PD nutrient solution, in full temperature concussion incubator 25 ℃, cultivate 96h under the 180r/min condition.After cultivation finishes, measure the thalline blastospore output in the PD nutrient solution with blood counting chamber, using the spore concentration of sterilized water adjustment bacterium liquid is 10
7Individual/mL is subsequent use.
On the Bechtop of sterilisable chamber, (spore concentration is 10 with the above-mentioned bacterium liquid that obtains with pipettor
7Individual/mL) quantitative being inoculated in the triangular flask that 100g solid fermentation substratum of the present invention is housed; Culture medium preparation is prepared according to the described method of embodiment 1-5, and each substratum carries out 4 groups of revision tests, and being divided into is 5 groups; As experimental group, inoculum size is 5mL bacterium liquid/bottle.After inoculation finishes; Gently shake triangular flask, make the abundant mixing of inoculation liquid and culture medium, then triangular flask is put into fermentation culture in the constant incubator; Culture temperature is 25 ℃; Be the visible mycelium that is covered with white on culture medium behind the cultivation 3-4d, behind the continuation cultivation 14d, carry out the mensuration of conidium output.
Control group adopts monomer substratum rice, takes by weighing a certain amount of rice, adds an amount of water rice is fully absorbed water, and takes by weighing rice that identical weight fully absorbs water and packs in the triangular flask 4 repetitions into.In temperature is 121 ℃, and pressure is high pressure moist heat sterilization 45min under the condition of 103.4205kPa, and behind the bacterium of having gone out, on the Bechtop of sterilisable chamber, (spore concentration of bacterium liquid is 10 with the quantitative inoculation of pipettor
7Individual/mL) to being equipped with in the rice solid medium triangular flask, inoculum size is similarly 5mL bacterium liquid/bottle.Triangular flask is put into fermentation culture in the constant incubator, and culture temperature is 25 ℃, and incubation time is 14d, carries out the mensuration of conidium output.
The mensuration of conidium output adopts the blood counting chamber assay method, and soon the matrix after the fermentation culture is diluted with sterilized water one by one, measures conidial output with blood counting chamber then.
The result finds to adopt its conidium mean yield of culture medium culturing Verticillium lecanii that other embodiment of the present invention prepare all 3.5 * 10
9Individual/more than the g.And in used solid fermentation substratum, contain: glucose 26gL
-1, yeast extract paste 25gL
-1, MgSO
40.14gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5 o'clock (according to embodiment 5 described method preparations), the conidium mean yield of mensuration has reached 7.5 * 10
9Individual/g.And the average out to as a result 2 * 10 that control group is measured
9Individual/g.
Know from above, use Verticillium lecanii solid medium of the present invention, more generally use cultivate as the solid-substrate fermentation of matrix with agricultural byproducts after, conidial output is significantly improved, conidial output has improved 1.75-3.75 doubly.And the present invention compares with monomer substratum rice with raw material as the carrier of substratum with agricultural-food waste material corn cob and greatly reduces production cost, when obtaining economic benefit, has reduced environmental pollution to a certain extent.
Claims (10)
1. Verticillium lecanii solid fermentation substratum is characterized in that described substratum is is that 1:2～1:5 is mixed and obtains after the sterilization by corn cob powder and nutritive medium by mass ratio;
Contain glucose 22～28gL in the described nutritive medium
-1, yeast extract paste 22～28gL
-1, MgSO
40.05～0.2gL
-1, K
2HP0
40.5～0.9gL
-1, MnSO
40.05-0.075gL
-1, FeSO
40.005-0.015gL
-1, KCl 0.25～0.4gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.0-7.0.
2. substratum as claimed in claim 1 is characterized in that described substratum is is that 1:3 is mixed and obtains after the sterilization by corn cob powder and nutritive medium by mass ratio.
3. according to claim 1 or claim 2 substratum is characterized in that containing in the described nutritive medium glucose 24～26gL
-1, yeast extract paste 24～26gL
-1, MgSO
40.1～0.15gL
-1, K
2HP0
40.6～0.8gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.3～0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5, and is preferred, contains glucose 26gL in the described nutritive medium
-1, yeast extract paste 25gL
-1, MgSO
40.14gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5.
4. substratum as claimed in claim 1 is characterized in that described corn cob powder is that the corn cob dried and crushed is become particle diameter is to obtain behind the particle of 2-4mm, and preferably the corn cob dried and crushed being become particle diameter is to obtain behind the particle of 3mm.
5. method for preparing the described substratum of claim 1 is characterized in that may further comprise the steps:
(1) raw materials pretreatment: corn cob is dried and with kibbler its pulverizing obtained the corn cob powder;
(2) proportion of raw materials: corn cob powder and nutritive medium are that 1:2～1:5 is mixed by mass ratio, contain glucose 22～28gL in the described nutritive medium
-1, yeast extract paste 22～28gL
-1, MgSO
40.05～0.2gL
-1, K
2HP0
40.5～0.9gL
-1, MnSO
40.05-0.075gL
-1, FeSO
40.005-0.015gL
-1, KCl 0.25～0.4gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.0-7.0;
(3) sterilization of substratum: the high pressure moist heat sterilization, obtaining with the corn cob is the Verticillium lecanii solid fermentation substratum of raw material and matrix.
6. method as claimed in claim 5 is characterized in that the corn cob powder described in the step (1) is that the corn cob dried and crushed is become particle diameter is to obtain behind the particle of 2-4mm, and preferably the corn cob dried and crushed being become particle diameter is to obtain behind the particle of 3mm.
7. method as claimed in claim 5 is characterized in that in the step (2) that being is that 1:3 is mixed with corn cob powder and nutritive medium by mass ratio.
8. method as claimed in claim 5 is characterized in that containing glucose 24～26gL in the nutritive medium described in the step (2)
-1, yeast extract paste 24～26gL
-1, MgSO
40.1～0.15gL
-1, K
2HP0
40.6～0.8gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.3～0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5, and is preferred, contains glucose 26gL in the described nutritive medium
-1, yeast extract paste 25gL
-1, MgSO
40.14gL
-1, K
2HP0
40.7gL
-1, MnSO
40.0625gL
-1, FeSO
40.01gL
-1, KCl 0.35gL
-1, the pH value of using NaOH solution to regulate nutritive medium is 6.5.
9. method as claimed in claim 5 is characterized in that the high pressure moist heat sterilization described in the step (3) is is 121 ℃ in temperature, and pressure is the 45-60min that sterilizes under the condition of 103.4205kPa.
10. each described substratum application in the Verticillium lecanii fermentation culture of claim 1-4.
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| CN102994483A (en) * | 2012-12-17 | 2013-03-27 | 福建农林大学 | Culture medium and method for producing chitinase by Lecanicillium |
| CN103013963A (en) * | 2012-12-17 | 2013-04-03 | 福建农林大学 | Culture medium and method for proteinase production through fermentation of Lecanicillium lecanii |
| US9144616B1 (en) | 2014-09-16 | 2015-09-29 | Green Products Company | Sanitized animal bedding material and process |
| CN105219692A (en) * | 2015-11-04 | 2016-01-06 | 中国农业大学 | Solid state fermentation produces the conidial method of Verticillium lecanii |
| CN106635948A (en) * | 2017-01-10 | 2017-05-10 | 广西大学 | Pollution-free preparing and culturing method for rice-false-smut-case thin-wall conidia |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994483A (en) * | 2012-12-17 | 2013-03-27 | 福建农林大学 | Culture medium and method for producing chitinase by Lecanicillium |
| CN103013963A (en) * | 2012-12-17 | 2013-04-03 | 福建农林大学 | Culture medium and method for proteinase production through fermentation of Lecanicillium lecanii |
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| US20160074543A1 (en) * | 2014-09-16 | 2016-03-17 | Green Products Company | Sanitized animal bedding material and process |
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| CN105219692A (en) * | 2015-11-04 | 2016-01-06 | 中国农业大学 | Solid state fermentation produces the conidial method of Verticillium lecanii |
| CN106635948A (en) * | 2017-01-10 | 2017-05-10 | 广西大学 | Pollution-free preparing and culturing method for rice-false-smut-case thin-wall conidia |
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