CN108041102A - A kind of method that diamondback moth biological agent is killed in fermented tcm compound material preparation - Google Patents
A kind of method that diamondback moth biological agent is killed in fermented tcm compound material preparation Download PDFInfo
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- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
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Abstract
The present invention relates to the methods that diamondback moth biological agent is killed in a kind of preparation of fermented tcm compound material, belong to technical field of biological fermentation.In order to solve the problems, such as that existing specific aim is poor, a kind of method that diamondback moth biological agent is killed in fermented tcm compound material preparation is provided, after being cleaned up including selection China tree, kuh-seng and tuber of stemona Chinese medicine material, after crushing processing, it adds in fermentation tank, adds water and be diluted to fermentation substrate；After sterilizing, the bacillus licheniformis of activation is according to 108Cfu/mL~109The amount of cfu/mL is inoculated into the fermentation substrate after sterilizing, carries out fermentation process, obtains zymotic fluid；Sediment is filtered out, obtains killing diamondback moth biological agent.It can realize the insecticidal function improved to diamondback moth in the biological agent that the present invention obtains containing toosendanin, matrine and Stemonidine and isostemonidine simultaneously, and there is the effect for promoting growth in itself to vegetable plant.
Description
Technical field
The present invention relates to the methods that diamondback moth biological agent is killed in a kind of preparation of fermented tcm compound material, belong to biofermentation
Technical field.
Background technology
Diamondback moth (Plutel la xylostel la) is lepidoptera diamond-back moth section insect, the small blue or green worm of alias, pointed at both ends, square
Block moth hangs corpse worm etc., belongs to the worldwide pest that migrates.Diamondback moth has generation more from generation to generation, fertility is strong, host range is wide,
Detection of insecticide resistance is high, it is difficult to the features such as preventing.Main wild cabbage of causing harm, violet cabbage, broccoli, a kind of sedge dish, leaf mustard, cauliflower, Chinese cabbage,
The crucifers such as rape, radish, and the annual whole world is up to 1,000,000,000 dollars for preventing the expense of diamondback moth.At present, for
The harm of diamondback moth mainly prevents diamondback moth using chemical pesticide, not only pollutes environment, also easily causes phyto toxicity, also
Easily cause person poultry poisoning.Therefore, the research and development of biological diamondback moth insecticide attract attention.
It is general that the deinsectization mode that desinsection is social recognition is carried out using chemical pesticide, still, have one using chemical pesticide
Fixed toxicity, the problem of being sprayed onto long-term existence toxic residue on plant.During vegetable cultivation, the use of insecticide is
It quite releases from sufferings, this will be because using various side effects caused by chemical pesticide, being unfavorable for the health of human body.As people give birth to
The protective awareness of dis environment and natural, ecological is increasingly enhanced the limit being subject to for the insecticide of chemical pesticide these high pesticide residues
System.Therefore, Pesticide use is reduced, substituting chemical pesticide with biological pesticide has become current pesticide development trend.
At present, the specific preferable insecticidal effect of the vegetable materials such as the China tree of discovery, kuh-seng and tuber of stemona, according to existing research
Situation, having developed China tree has the effect of desinsection, and toosendanin therein has very strong stomach poison function to cabbage caterpillar etc., can break
Bad midgut tissue, block nerves conduct and cause pest paralysis, stupor, death.And kuh-seng contains many secondary metabolites, it is bitter
Ginseng ether extract and alcohol extract have staphylococcus aureus stronger bacteriostasis；Kuh-seng flooding agent to trichophyton, with one heart
Property trichophyta, Trichophyton schoenleini, Du small gemma tinea bacterium of big belly difficult to understand etc. have inhibitory action.Matrine can benumb nerve center, make polypide
Protein coagulating closes polypide stomata, pest is made to be choked to death；The tuber of stemona is containing stemonine, Stemonidine, isostemonidine, original hundred
A variety of secondary metabolites such as portion's alkali, stenine alkali, sinostemonine tuberostemonine, hordorine, sessilistemonine, to mosquitos and flies
Larva, head louse, crab louse, clothing lice and bedbug etc. have killing effect, and the high concentration tuber of stemona plays the role of killing mouse pinworm in vitro.Cause
This, also produces the insecticide of biological agent some researches show that the mode by using fermentation at present.As Chinese patent application is (public
The number of opening：CN106867947A a kind of method for preparing insecticide through microbial fermentation using China tree as raw material, this method bag) are disclosed
It includes that bacillus amyloliquefaciens, bacillus pumilus, Dipel are inoculated in respectively in seed culture medium cultivate and survives
Mixed Microbes are made into proportion, then by China tree after ethyl alcohol and water process, are put into after crushing and water-adding, sterilizing in fermentation tank, it will be upper
Mixed Microbes inoculation is stated, and after 37 DEG C are cultivated 3-12 days, obtains corresponding zymotic fluid, after filtering, obtained zymotic fluid can kill
Worm agent.Although can avoid the problem that inactivation and organic solvent pollution to a certain extent by way of biofermentation,
It is to ferment in a manner that a variety of biological bacterias coexist, there are of high cost, the problems such as the complicated condition that a variety of bacterium coexist,
And it is only the fermented and cultured for single plant.
The content of the invention
The present invention provides a kind of fermented tcm compound material preparation and kills pickles for above defect in the prior art
The method of moth biological agent, solve the problems, such as be how to realize have both improve plant growth and high activity kill diamondback moth worm property
Energy.
The purpose of the present invention is what is be achieved by the following technical programs, a kind of fermented tcm compound material preparation is killed small
The method of diamond-back moth biological agent, which is characterized in that this method comprises the following steps：
A, after selection China tree, kuh-seng and tuber of stemona Chinese medicine material clean up, after crushing processing, add in fermentation tank,
It adds water and is diluted to fermentation substrate；
B, after obtained fermentation substrate being carried out sterilization treatment, then by activated bacillus licheniformis according to 108cfu/
ML~109The amount of cfu/mL is inoculated into the fermentation substrate after sterilizing, and fermentation temperature is made to be sent out under conditions of 25 DEG C~35 DEG C
Ferment processing, obtains zymotic fluid；
C, obtained zymotic fluid is filtered removing sediment, obtains killing diamondback moth biological agent.
By using single bacillus licheniformis as microorganism, allowing to China tree, kuh-seng and the tuber of stemona three kinds of Chinese medicines
Mixed raw material carries out fermented and cultured as substrate simultaneously, can effectively avoid coexisting and generating phase for a variety of superior microorganism bacterial strains
Mutually the problem of competition, and fermented and cultured is carried out using single bacillus licheniformis, allow to preferably utilize fermentation substrate
Nutriment can be such that the active ingredient in China tree, kuh-seng and the tuber of stemona is effectively released, so as to the fermentation made
The function that can be effectively killed when liquid is as biological insecticides to diamondback moth, and bacillus licheniformis has growth-promoting in itself
Effect can make, using having plant such as vegetables etc. during this desinsection biological agent the effect for promoting growth, to improve the life of plant
Long ability；Meanwhile can also ensure that the activity of active material in zymotic fluid by way of fermentation, increase it and kill the work(of diamondback moth
Energy.
In the method for diamondback moth biological agent is killed in the preparation of above-mentioned fermented tcm compound material, preferably, institute in step A
State China tree：Kuh-seng：The tuber of stemona：The mass ratio of water is 1：0.5~0.7：0.7~0.9：10~15.In order to make by being obtained after fermentation
To zymotic fluid in each active material ingredient to diamondback moth have better killing ability, especially make the river obtained from China tree
Chinaberry element and the matrine obtained from kuh-seng occupy higher ratio, and improve has better block nerves and paralysis to diamondback moth
The effect of nerve.
In the method for diamondback moth biological agent is killed in the preparation of above-mentioned fermented tcm compound material, preferably, institute in step A
Pulverization process is stated added with ice powder, the additive amount of the ice powder is the 2.0%~5.0% of China tree quality.Since ice powder has one
Fixed hardness and corner angle can play destruction, so that preferably releasing during crushing to plant tissue cell's wall
Put intracellular active ingredient；Meanwhile, it is capable to which it is more thorough to crush plant tissue, improving bacillus licheniformis has them
It imitates and utilizes, and the ice powder used dissolves the rear directly formation certain zymotic fluid raw material of water, its side effect will not be generated to raw material.Make
To be further preferred, the temperature of the ice powder is -30 DEG C~-10 DEG C.Improve ice powder hardness, make have preferably puncture energy
Power.
In the method for diamondback moth biological agent is killed in the preparation of above-mentioned fermented tcm compound material, preferably, institute in step B
It states and carbon source is added into the backward fermentation substrate of sterilizing, the additive amount of the carbon source is the 5%~10% of the addition of China tree.Pass through
A small amount of carbon source is added in early period, the growth and breeding of bacillus licheniformis, a large amount of lichens gemma bars of flourish can be improved
Bacterium more fully can add China tree, kuh-seng and tuber of stemona raw material with quick utilize.As further preferred, the carbon source choosing
From one or more of glucose, fructose and sucrose.Be conducive to improve the utilization ratio of bacillus licheniformis.
In the method for diamondback moth biological agent is killed in the preparation of above-mentioned fermented tcm compound material, preferably, institute in step B
The time for stating fermentation process is 7~10 days.Purpose is to sufficiently utilize the nutritional ingredient of fermentation substrate, improves plant tissue
The release of active ingredient in cell.
In the method for diamondback moth biological agent is killed in the preparation of above-mentioned fermented tcm compound material, preferably, institute in step B
The pH value of system is 6.5~7.5 when stating fermentation process.The environment of fermented and cultured is made to be more advantageous to the growth of bacillus licheniformis.
In the method for diamondback moth biological agent is killed in the preparation of above-mentioned fermented tcm compound material, preferably, institute in step C
It states to kill and contains toosendanin, matrine, Stemonidine and isostemonidine alkali composition in diamondback moth biological agent.By using the present invention
Bacillus licheniformis as Promoting bacteria carry out fermented and cultured, the effective active composition river in China tree, kuh-seng and the tuber of stemona can be made
Chinaberry element, matrine, Stemonidine and isostemonidine preferably discharge, so as to which the zymotic fluid made has preferably diamondback moth
Killing ability, and with effect with strong points.
In conclusion it compared with prior art, the present invention has the following advantages：
The present invention is allowed to simultaneously by fermenting using single bacillus licheniformis as unique microbial bacteria to hardship
Chinaberry, kuh-seng and the tuber of stemona carry out disposable fermented and cultured, and are effectively grown using them as nutritional ingredient, so that having
Beneficial to the release of active ingredient therein, make can simultaneously to contain in biological agent toosendanin, matrine and Stemonidine and different
Stemonidine realization improves the insecticidal function to diamondback moth, and the bacillus licheniformis used has rush in itself to vegetable plant
Into the effect of growth, there is the effect for promoting growth while this biological agent is used to vegetable plant.
Specific embodiment
Below by specific embodiment, the technical solutions of the present invention will be further described, but the present invention is simultaneously
It is not limited to these embodiments.
Embodiment 1
It is former that usage ratio according to China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine material chooses respective Chinese medicine
Material first chooses the fresh China tree Chinese medicine material of healthy growth, and the dry weight of China tree is made after being cleaned up with clear water, then to be adopted for 900g
Crushed with pulverizer and be processed into China tree slurries, can be added in crushing process a small amount of water destroyed to avoid overheat activity into
Point, using fresh China tree per se with a part of water, also for playing the effect that prevents overheat；Fresh kuh-seng is chosen again
Chinese medicine material makes the dry weight of kuh-seng for 700g, after being cleaned up with clear water, then is crushed using pulverizer and is processed into kuh-seng slurry
Liquid finally chooses fresh tuber of stemona Chinese medicine material again, and the dry weight of the tuber of stemona is made after cleaning up, then to crush place for 630g
Tuber of stemona slurries are managed into, then, three kinds of slurries obtained above is added in fermentation tank and are mixed, water is added and is diluted to 10L
The aqueous fermentation substrate of volume；By obtained fermentation substrate carry out sterilization treatment after, the temperature that sterilization treatment uses for 80 DEG C,
The time of sterilization treatment is 15 minutes, after sterilization treatment, then by activated bacillus licheniformis according to 109The amount of cfu/mL connects
Fermentation substrate after kind to sterilizing, directly by being commercially available, specific activity uses conventional bacillus licheniformis here
Seed culture medium carry out culture activation process, obtain the bacillus licheniformis liquid containing activation, specific seed culture based formulas
Ingredient is as follows：Peptone：10g/L；Enzyme mother's cream：5g/L；Sodium chloride：10g/L, remaining is water, and obtained culture solution is taken centainly
Amount makes in the solution that inoculum concentration reaches the above-mentioned fermentation substrate of requirement addition, after the completion of inoculation, controls the fermentation of fermentation system
Temperature controls the pH value of system and fermentation process 10 is carried out in the range of 7.1~7.3 under conditions of 25 DEG C~30 DEG C
My god, after fermentation, obtain corresponding zymotic fluid；Obtained zymotic fluid progress filtering element (cartridge) is filtered to remove sediment again, you can
To killing diamondback moth biological agent.Obtained biological agent is analyzed, concrete analysis result is as follows：
It is higher containing toosendanin, matrine, Stemonidine and isostemonidine alkali composition in this biological agent, and toosendanin：
Matrine：Stemonidine：The content of isostemonidine is frequently 1：0.5：0.2：0.4.It can also be analyzed from content situation,
The recovery rate of toosendanin is up to more than 60% in middle China tree, and the extraction rate reached of matrine is to more than 40% in kuh-seng, hundred in the tuber of stemona
Portion determines alkali and total extraction of isostemonidine reaches more than 50%.
The biology obtained using the present invention carries out killing diamondback moth experiment, and the vegetable plant endangered by diamondback moth is used this life
Object preparation (directly adopting obtained liquid bio preparation after being diluted with water to 100 times) carries out sprinkling desinsection processing.It was found that sprinkling
, can be by the substantially all killing of diamondback moth in two days after this biological agent, killing rate reaches more than 95%, five days or more bases
It can be killed completely on this, there is the effect for killing diamondback moth well；And after spraying, the growing state of vegetable is more preferable, vegetable tool
The vegetable of the greenery such as selecting green vegetables, it is seen that there is certain growth-promoting functions effect.
Embodiment 2
It is former that usage ratio according to China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine material chooses respective Chinese medicine
Material first chooses the fresh China tree Chinese medicine material of healthy growth, after being cleaned up with clear water, China tree first is cut into small particle,
It is crushed again using pulverizer and is processed into China tree slurries；Fresh kuh-seng Chinese medicine material is chosen again, after being cleaned up with clear water,
It is crushed again using pulverizer and is processed into kuh-seng slurries, finally choose fresh tuber of stemona Chinese medicine material again, after cleaning up,
It crushes again and is processed into tuber of stemona slurries, then, three kinds of slurries obtained above are added in fermentation tank and are mixed, are added
Water is diluted to aqueous fermentation substrate；The addition of each ingredient is according to China tree：Kuh-seng：The tuber of stemona：The mass ratio of water is 1：0.5：
0.7：10, here the quality of China tree, kuh-seng and the tuber of stemona converted according to dry weight, by obtained fermentation substrate carry out sterilization treatment
Afterwards, for the temperature that sterilization treatment uses for 85 DEG C, time of sterilization treatment is 10 minutes, after sterilization treatment, then by activated ground
Clothing bacillus is according to 108The amount of cfu/mL is inoculated into the fermentation substrate after sterilizing, and bacillus licheniformis directly passes through purchase here
It can buy, specific activity carries out culture activation process using conventional seed culture medium, obtains the lichens bud containing activation
Spore bacillus liquid, the activation of the bacillus licheniformis of the present embodiment after the completion of inoculation, control fermentation system with the consistent of embodiment 1
Fermentation temperature under conditions of 30 DEG C~35 DEG C, and make system pH value control carried out in the range of 6.5~7.0 at fermentation
Reason 7 days, after fermentation, obtains corresponding zymotic fluid；Coarse filtration is first carried out, removes solid residue larger in zymotic fluid, then
Carry out refined filtration, you can obtain killing diamondback moth biological agent.
It is higher containing toosendanin, matrine, Stemonidine and isostemonidine alkali composition in this biological agent, and toosendanin：
Matrine：Stemonidine：The content of isostemonidine is frequently 1：0.4：0.3：0.5.It can also be analyzed from content situation,
The recovery rate of toosendanin is up to more than 60% in middle China tree, and the extraction rate reached of matrine is to more than 40% in kuh-seng, hundred in the tuber of stemona
Portion determines alkali and total extraction of isostemonidine reaches more than 50%.
The biology obtained using the present invention carries out killing diamondback moth experiment, and the vegetable plant endangered by diamondback moth is used this life
Object preparation (directly adopting obtained liquid bio preparation after being diluted with water to 200 times) carries out sprinkling desinsection processing.It was found that sprinkling
, can be by the substantially all killing of diamondback moth in two days after this biological agent, killing rate reaches more than 93%, five days or more bases
It can be killed completely on this, there is the effect for killing diamondback moth well, and after sprinkling, the growing state of vegetable is more preferable, vegetable tool
The vegetable of the greenery such as selecting green vegetables, it is seen that there is certain growth-promoting functions effect.
Embodiment 3
It is former that usage ratio according to China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine material chooses respective Chinese medicine
Material first chooses the fresh China tree Chinese medicine material of healthy growth, after being cleaned up with clear water, China tree first is cut into small particle,
It is crushed again using pulverizer and is processed into China tree slurries；Fresh kuh-seng Chinese medicine material is chosen again, after being cleaned up with clear water,
It is crushed again using pulverizer and is processed into kuh-seng slurries, finally choose fresh tuber of stemona Chinese medicine material again, after cleaning up,
It crushes again and is processed into tuber of stemona slurries, then, three kinds of slurries obtained above are added in fermentation tank and are mixed, are added
Water is diluted to aqueous fermentation substrate；The addition of each ingredient is according to China tree：Kuh-seng：The tuber of stemona：The mass ratio of water is 1：0.7：
0.9：15, here the quality of China tree, kuh-seng and the tuber of stemona converted according to dry weight, by obtained fermentation substrate carry out sterilization treatment
Afterwards, for the temperature that sterilization treatment uses for 80 DEG C, the time of sterilization treatment is 15 minutes, and after sterilization treatment, choosing adds in sucrose, makes sugarcane
The addition of sugar adds in the 5% of weight for China tree, is preferably converted according to the dry weight of China tree, then by activated lichens gemma
Bacillus is according to 1.5x 108The amount of cfu/mL is inoculated into the fermentation substrate after sterilizing, and bacillus licheniformis directly passes through purchase here
It obtains, after the completion of inoculation, controls the fermentation temperature of fermentation system under conditions of 28 DEG C~32 DEG C, and make the pH value of system
Control carries out fermentation process 8 days in the range of 7.1~7.5, after fermentation, obtains corresponding zymotic fluid；Coarse filtration is first carried out,
Solid residue larger in zymotic fluid is removed, then carries out refined filtration, you can obtains killing diamondback moth biological agent.
It is higher containing toosendanin, matrine, Stemonidine and isostemonidine alkali composition in this biological agent, and toosendanin：
Matrine：Stemonidine：The content of isostemonidine is frequently 1：0.4：0.3：0.5.It can also be analyzed from content situation,
The recovery rate of toosendanin is up to more than 60% in middle China tree, and the extraction rate reached of matrine is to more than 40% in kuh-seng, hundred in the tuber of stemona
Portion determines alkali and total extraction of isostemonidine reaches more than 50%.
The biology obtained using the present invention carries out killing diamondback moth experiment, and the vegetable plant endangered by diamondback moth is used this life
Object preparation (directly adopting obtained liquid bio preparation after being diluted with water to 200 times) carries out sprinkling desinsection processing.It was found that sprinkling
, can be by the substantially all killing of diamondback moth in two days after this biological agent, killing rate reaches more than 93%, five days or more bases
It can be killed completely on this, there is the effect for killing diamondback moth well, and after sprinkling, the growing state of vegetable is more preferable, vegetable tool
The vegetable of the greenery such as selecting green vegetables, it is seen that there is certain growth-promoting functions effect.
Embodiment 4
China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine respectively after clear water cleaning treatment is clean is former
Material chooses respective Chinese medicine material according to the usage ratio of China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine material, will
Three kinds of Chinese medicine material choosings are shredded into small particle, add ice powder, and the temperature for the ice powder being initially added is -30 DEG C, makes ice
The addition of powder is the 5.0% of China tree dry weight, then carries out strong stirring pulverization process, and mixing speed is controlled at 10000 revs/min
Clock or so obtains direct mixed mixed slurry, adds water and be diluted to aqueous fermentation substrate；Make the addition of each ingredient
According to China tree：Kuh-seng：The tuber of stemona：The mass ratio of water is 1：0.6：0.7：12, the quality of China tree, kuh-seng and the tuber of stemona is according to dry weight here
Converted, by obtained fermentation substrate carry out sterilization treatment after, the temperature that sterilization treatment uses for 80 DEG C, sterilization treatment when
Between for 20 minutes, after sterilization treatment, choosing adds in glucose, and the addition of glucose is made to add in the 10% of weight for China tree, best
It is converted according to the dry weight of China tree, then by activated bacillus licheniformis according to 1.2x 108The amount of cfu/mL, which is inoculated into, goes out
Fermentation substrate after bacterium, here bacillus licheniformis correspond generally to activated lichens bud directly by being commercially available
Spore bacillus culture solution 100mL or so after the completion of inoculation, controls the fermentation temperature of fermentation system under conditions of 30 DEG C~33 DEG C,
And control the pH value of system and fermentation process is carried out in the range of 7.1~7.5 9 days, after fermentation, fermented accordingly
Liquid；Coarse filtration is first carried out, solid residue larger in zymotic fluid is removed, then carries out refined filtration, you can obtains killing diamondback moth biology system
Agent.
It is higher containing toosendanin, matrine, Stemonidine and isostemonidine alkali composition in this biological agent, and toosendanin：
Matrine：Stemonidine：The content of isostemonidine is frequently 1：0.6：0.4：0.6.It can also be analyzed from content situation,
The recovery rate of toosendanin is up to more than 75% in middle China tree, and the extraction rate reached of matrine is to more than 60% in kuh-seng, hundred in the tuber of stemona
Portion determines alkali and total extraction of isostemonidine reaches more than 60%.
The biology obtained using the present invention carries out killing diamondback moth experiment, and the vegetable plant endangered by diamondback moth is used this life
Object preparation (directly adopting obtained liquid bio preparation after being diluted with water to 200 times) carries out sprinkling desinsection processing.It was found that sprinkling
, can be by the substantially all killing of diamondback moth in two days after this biological agent, killing rate reaches more than 93%, five days or more bases
It can be killed completely on this, there is the effect for killing diamondback moth well, and after sprinkling, the growing state of vegetable is more preferable, vegetable tool
The vegetable of the greenery such as selecting green vegetables, it is seen that there is certain growth-promoting functions effect.
Embodiment 5
China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine respectively after clear water cleaning treatment is clean is former
Material chooses respective Chinese medicine material according to the usage ratio of China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine material, will
Three kinds of Chinese medicine material choosings are shredded into small particle, add ice powder, and the temperature for the ice powder being initially added is -10 DEG C, makes ice
The addition of powder is the 2.0% of China tree dry weight, then carries out strong stirring pulverization process, and mixing speed is controlled at 8000 revs/min
Clock or so obtains direct mixed mixed slurry, adds water and be diluted to aqueous fermentation substrate；Make the addition of each ingredient
According to China tree：Kuh-seng：The tuber of stemona：The mass ratio of water is 1：0.6：0.8：13, the quality of China tree, kuh-seng and the tuber of stemona is according to dry weight here
Converted, by obtained fermentation substrate carry out sterilization treatment after, the temperature that sterilization treatment uses for 90 DEG C, sterilization treatment when
Between for 10 minutes, after sterilization treatment, choosing adds in sucrose, the addition of sucrose is made to add in the 8% of weight for China tree, preferably according to hardship
The dry weight of chinaberry is converted, then by activated bacillus licheniformis according to 3.0x 108After the amount of cfu/mL is inoculated into sterilizing
Fermentation substrate, here bacillus licheniformis correspond generally to activated bacillus licheniformis directly by being commercially available
Culture solution 100mL or so after the completion of inoculation, controls the fermentation temperature of fermentation system under conditions of 30 DEG C~33 DEG C, and makes body
The pH value of system is controlled carries out fermentation process 9 days in the range of 7.1~7.5, after fermentation, obtains corresponding zymotic fluid；First
Coarse filtration is carried out, solid residue larger in zymotic fluid is removed, then carries out refined filtration, you can obtain killing diamondback moth biological agent.
It is higher containing toosendanin, matrine, Stemonidine and isostemonidine alkali composition in this biological agent, and toosendanin：
Matrine：Stemonidine：The content of isostemonidine is frequently 1：0.5：0.4：0.5.It can also be analyzed from content situation,
The recovery rate of toosendanin is up to more than 75% in middle China tree, and the extraction rate reached of matrine is to more than 60% in kuh-seng, hundred in the tuber of stemona
Portion determines alkali and total extraction of isostemonidine reaches more than 60%.
The biology obtained using the present invention carries out killing diamondback moth experiment, and the vegetable plant endangered by diamondback moth is used this life
Object preparation (directly adopting obtained liquid bio preparation after being diluted with water to 500 times) carries out sprinkling desinsection processing.It was found that sprinkling
, can be by the substantially all killing of diamondback moth in two days after this biological agent, killing rate reaches more than 90%, five days or more bases
It can be killed completely on this, there is the effect for killing diamondback moth well, and after sprinkling, the growing state of vegetable is more preferable, vegetable tool
The vegetable of the greenery such as selecting green vegetables, it is seen that there is certain growth-promoting functions effect.
Comparative example 1
This comparative example selects single Dipel to carry out specific ratio implementation, Dipel as microbial bacteria
Pass through commercially available purchase.
It is former that usage ratio according to China tree Chinese medicine material, kuh-seng Chinese medicine material and tuber of stemona Chinese medicine material chooses respective Chinese medicine
Material first chooses the fresh China tree Chinese medicine material of healthy growth, and the dry weight of China tree is made after being cleaned up with clear water, then to be adopted for 900g
Crushed with pulverizer and be processed into China tree slurries, can be added in crushing process a small amount of water destroyed to avoid overheat activity into
Point, using fresh China tree per se with a part of water, also for playing the effect that prevents overheat；Fresh kuh-seng is chosen again
Chinese medicine material makes the dry weight of kuh-seng for 700g, after being cleaned up with clear water, then is crushed using pulverizer and is processed into kuh-seng slurry
Liquid finally chooses fresh tuber of stemona Chinese medicine material again, and the dry weight of the tuber of stemona is made after cleaning up, then to crush place for 630g
Tuber of stemona slurries are managed into, then, three kinds of slurries obtained above is added in fermentation tank and are mixed, water is added and is diluted to 10L
The aqueous fermentation substrate of volume；By obtained fermentation substrate carry out sterilization treatment after, the temperature that sterilization treatment uses for 80 DEG C,
The time of sterilization treatment is 15 minutes, after sterilization treatment, then by activated Dipel according to 109The amount inoculation of cfu/mL
Fermentation substrate after to sterilizing, specific activity carry out culture activation process using conventional seed culture medium, obtain containing activation
Bacillus licheniformis liquid, specific seed culture medium formula components are as follows：Peptone：10g/L；Enzyme mother's cream：5g/L；Chlorination
Sodium：10g/L, remaining is water, and obtained culture solution, which is taken, a certain amount of to be made inoculum concentration reach requirement to add in the molten of above-mentioned fermentation substrate
In liquid, after the completion of inoculation, the fermentation temperature of fermentation system is controlled under conditions of 25 DEG C~30 DEG C, and makes the pH value of system
Control carries out fermentation process 10 days in the range of 7.1~7.3, after fermentation, obtains corresponding zymotic fluid；It will obtain again
Zymotic fluid carries out filtering element (cartridge) and is filtered to remove sediment, you can obtains killing diamondback moth biological agent.Obtained biological agent is divided
Analysis, concrete analysis result are as follows：
It is higher containing toosendanin, matrine, Stemonidine and isostemonidine alkali composition in this biological agent, and toosendanin：
Matrine：Stemonidine：The content of isostemonidine is frequently 1：0.1：0.05：0.1, content ratio is quite larger, from content
Situation can also analyze, and the recovery rate of toosendanin is up to more than 40% wherein in China tree, the extraction rate reached of matrine in kuh-seng
To more than 10%, in the tuber of stemona total extraction of Stemonidine and isostemonidine reach more than 15%.
The biology obtained using the present invention carries out killing diamondback moth experiment, and the vegetable plant endangered by diamondback moth is used this life
Object preparation (directly adopting obtained liquid bio preparation after being diluted with water to 100 times) carries out sprinkling desinsection processing.It was found that sprinkling
, can be by the substantially all killing of diamondback moth in two days after this biological agent, killing rate reaches more than 70%, five days or more bases
It is also merely able to reach 85% or so in sheet, the effect for killing diamondback moth is relatively poor；And after spraying, the growing state of vegetable is general,
The vegetable of the greenery such as vegetable tool selecting green vegetables does not promote the effect of growth significantly.
Comparative example 2
This comparative example selects single bacillus pumilus to carry out specific ratio implementation, short and small gemma as microbial bacteria
For bacillus by commercially available purchase, specific embodiment is consistent with comparative example 1, differs only in using bacillus pumilus generation
For Dipel.Obtain corresponding biological agent.
It is higher containing toosendanin, matrine, Stemonidine and isostemonidine alkali composition in this biological agent, and toosendanin：
Matrine：Stemonidine：The content of isostemonidine is frequently 1：0.2：0.1：0.1, content ratio is quite larger, from content feelings
Condition can also analyze, and the recovery rate of toosendanin is up to more than 45% wherein in China tree, and the extraction rate reached of matrine arrives in kuh-seng
More than 15%, in the tuber of stemona total extraction of Stemonidine and isostemonidine reach more than 15%.
The biology obtained using the present invention carries out killing diamondback moth experiment, and the vegetable plant endangered by diamondback moth is used this life
Object preparation (directly adopting obtained liquid bio preparation after being diluted with water to 100 times) carries out sprinkling desinsection processing.It was found that sprinkling
, can be by the substantially all killing of diamondback moth in two days after this biological agent, killing rate reaches more than 75%, five days or more bases
It is also merely able to reach 85% or so in sheet, the effect for killing diamondback moth is relatively poor；And after spraying, the growing state of vegetable is general,
The vegetable of the greenery such as vegetable tool selecting green vegetables does not promote the effect of growth significantly.
Specific embodiment described in the present invention is only to spirit explanation for example of the invention.Technology belonging to the present invention is led
The technical staff in domain can do various modifications or additions to described specific embodiment or replace in a similar way
Generation, but without departing from spirit of the invention or beyond the scope of the appended claims.
It is skilled to this field although having been made a detailed description to the present invention and being cited some specific embodiments
For technical staff, as long as it is obvious that can make various changes or correct without departing from the spirit and scope of the present invention.
Claims (9)
1. the method that diamondback moth biological agent is killed in a kind of preparation of fermented tcm compound material, which is characterized in that this method include with
Lower step：
A, after selection China tree, kuh-seng and tuber of stemona Chinese medicine material clean up, after crushing processing, add in fermentation tank, then add
Water is diluted to fermentation substrate；
B, after obtained fermentation substrate being carried out sterilization treatment, then by activated bacillus licheniformis according to 108Cfu/mL~
109The amount of cfu/mL is inoculated into the fermentation substrate after sterilizing, and fermentation temperature is made to be carried out under conditions of 25 DEG C~35 DEG C at fermentation
Reason, obtains zymotic fluid；
C, obtained zymotic fluid is filtered removing sediment, obtains killing diamondback moth biological agent.
2. the method that diamondback moth biological agent is killed in the preparation of fermented tcm compound material according to claim 1, which is characterized in that
China tree described in step A：Kuh-seng：The tuber of stemona：The mass ratio of water is 1：0.5~0.7：0.7~0.9：10~15.
3. the method that diamondback moth biological agent is killed in the preparation of fermented tcm compound material according to claim 1, which is characterized in that
Pulverization process described in step A is added with ice powder, and the additive amount of the ice powder is the 2.0%~5.0% of China tree quality.
4. the method that diamondback moth biological agent is killed in the preparation of fermented tcm compound material according to claim 3, which is characterized in that
The temperature of the ice powder is -30 DEG C~-10 DEG C.
5. the method that diamondback moth biological agent is killed in the preparation of fermented tcm compound material according to claim 1, which is characterized in that
Be added into carbon source in the backward fermentation substrate of sterilizing described in step B, the additive amount of the carbon source for the addition of China tree 5%~
10%.
6. the method that diamondback moth biological agent is killed in the preparation of fermented tcm compound material according to claim 5, which is characterized in that
The carbon source is selected from one or more of glucose, fructose and sucrose.
7. the method for killing diamondback moth biological agent is prepared according to fermented tcm compound material described in claim 1-6 any one,
It is characterized in that, the time of fermentation process described in step B is 7~10 days.
8. the method for killing diamondback moth biological agent is prepared according to fermented tcm compound material described in claim 1-6 any one,
It is characterized in that, the pH value of system is 6.5~7.5 during fermentation process described in step B.
9. the method for killing diamondback moth biological agent is prepared according to fermented tcm compound material described in claim 1-6 any one,
Contain toosendanin, matrine, Stemonidine and isostemonidine in diamondback moth biological agent it is characterized in that, being killed described in step C
Alkali composition.
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