CN102258059B - Method for preparing ralstonia solanacearum-resistant bacillus subtilis microbial inoculum - Google Patents
Method for preparing ralstonia solanacearum-resistant bacillus subtilis microbial inoculum Download PDFInfo
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Abstract
The invention discloses a method for preparing a ralstonia solanacearum-resistant bacillus subtilis microbial inoculum. The method comprises strain activation, shaking table amplification culture, seeding tank amplification culture, fermenter fermentation, preparation of the microbial inoculum and the like. The method has the advantages that: in the fermenting process of a strain, an organic nitrogen source is supplemented at a later period, so that the strain undergoes metabolic growth, more basic protein antibiotics are generated, normal generation of germs is facilitated simultaneously, and an efficient ralstonia solanacearum-resistant bacillus subtilis microbial inoculum is obtained finally.
Description
Technical field
The present invention relates to a kind of microbial inoculum of anti-tobacco bacterial wilt, specifically a kind of preparation method of bacillus subtilis microbial agent of anti-tobacco bacterial wilt belongs to the bacteria agent field.
Background technology
Tobacco bacterial wilt is a kind of bacteriosis, generally occurs in each cigarette district, and usually outbreak of epidemic causes crushing loss.This disease pathogeny: Ralstonia solanacearum and blue or green withered false pseudomonas bacillus, can infect 200 various plants of more than 30 sections.More obvious to tobacco, tomato, peanut and potato infringement.The rhizome disease mixed occurrences such as normal and balck shank endanger more serious.
Bacillus subtilis (
Bacillus subtilis) extensively being present in occurring in nature, this bacterium is as one of biocontrol of plant disease bacterium, because this bacterium can produce heat-resisting gemma, namely is easy to produce, the processing of microbial inoculum, is easy to again survival, field planting and breeding.Mass production processes is simple, cost is also lower, use conveniently, storage life is long, and the compatibility of the stability of its microbial inoculum and chemical pesticide and all be better than asporogenic bacterium and fungi with the uniformity aspect of different plant different year control efficiency, be a kind of comparatively ideal Biocontrol microorganism.
Summary of the invention
The object of the invention is to, a kind of preparation method who the tobacco bacterial wilt evil is had the microorganism bacillus subtilis agent of efficient, nontoxic, safety and environmentally safe is provided.
In the fermentation of bacillus later stage, control, the functional organic nitrogen source of interpolation allotment by reasonable adjusting fermenting and producing parameter, impel bacillus in later stage metabolic growth, produce the metabolism of basic protein, again by adjusting zymotechnique control parameter, impel thalline to form gemma, when the thalline spore forming rate reaches 55～65% when above, no longer cook the adjustment of zymotic fluid pH, allow it naturally change, when the zymotic fluid thalline forms the gemma rate more than 85%, final zymotic fluid pH value is increased at 7.7～8.0 o'clock, fermentation ends naturally.
Technical scheme of the present invention:
A kind of preparation method of bacillus subtilis microbial agent of anti-tobacco bacterial wilt may further comprise the steps:
1, actication of culture: the bacillus subtilis to preservation carries out activation culture to bacillus exfoliation;
Activating used culture medium prescription is: beef peptone 3g, and peptone 5g, agar 16g, water is settled to 1000ml; Be used for the activation of bacillus subtilis and the mensuration of viable count;
2, shaking table enlarges cultivation: the bacillus subtilis after will activating is inoculated into and carries out shaking table expansion cultivation in the liquid nutrient medium.Initial pH value is 7.0～7.2, and cultivation temperature is 30 ℃, and shaking speed is 200rpm, till 15～20% bacillus exfoliation;
The prescription of used liquid nutrient medium is: corn flour 6.5g, glucose 2.5g, beancake powder 10.1g, fish meal 2.5g, CaCO
33.5g, (NH
4)
2SO
40.5g, K
2HPO
40.15g, MgSO
47H
2O 0.1g, MnSO
4H
2O 0.1g, water is settled to 500ml.
3, seeding tank enlarges cultivation: under sterile working, the bacterium liquid that obtains is cultivated in shaking table expansion to be linked in the seeding tank that contains the seed tank culture base, initial stage ventilating ratio 1 ︰ 0.25, namely one cube of zymotic fluid one minute ventilation is 0.25 cube, later stage ventilating ratio 1 ︰ 1; Simultaneously, the pH value of controlled fermentation liquid is 6.9～7.3, and temperature is 32 ℃, and the speed of agitator of fermentation tank is 80～220 rpm; Enter the later stage of exponential phase to bacillus;
In time carry out to the inspection of seed fermentation liquid with to the observation of bacillus upgrowth situation; The 12h thalli growth is in exponential growth mid-term behind the bacterial classification inoculation; When bacillus enters later stage of exponential phase, carry out aseptic transferred species access fermentation tank, ferment;
Used seed tank culture based formulas is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml.
4, ferment tank
Change the seeding tank bacterium liquid that is cultured to later stage exponential phase of growth over to fermentation tank that fermentation tank culture medium is housed under aseptic condition, canned amount is 70% of fermentation tank volume, carries out the early stage of fermentation tank and cultivates and the later stage cultivation;
(1) fermentation tank cultural method in early stage is: to the fermentation tank of access seed liquor, 28～32 ℃ of temperature, pH6.9～7.3, ventilation 1 ︰ 0.25～1.1, speed of agitator 150～180 rpm; Keep zymotic fluid total sugar content 0.35～0.6%, total nitrogen content 30～45ng/ml, be cultured to spore forming rate 55～65%, enter the fermentation later stage;
Fermentation tank will in time check and observe the growth situation between culture period early stage, growth along with bacterial classification, the increase of cell density, strengthen gradually ventilation, increase dissolved oxygen, improve rotating speed, to cooperate the oxygen demand of growth, simultaneously, keep zymotic fluid pH value by the aseptic sealant supplement cylinder that links to each other with fermentation tank;
(2) cultural method is the fermentation tank later stage: no longer regulate zymotic fluid pH value, use organic nitrogen source to regulate the total nitrogen content of zymotic fluid, the control total nitrogen content is cultured to spore forming rate 80% or more, pH value more than 7.7, the later stage fermentation end more than 40ng/ml;
The prescription of described fermentation tank culture medium is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
The preparation method of described organic nitrogen source is: be that the ratio of 1 ︰, 1 ︰, 1 ︰, 1 ︰, 1 ︰ 1 is mixed with groundnut meal, soybean cake powder, cotton seed powder cake, dusty yeast, dried silkworm chrysalis meal, fish meal according to weight ratio, be modulated into weight fraction and be 9%～16% mixed solution, join in the reactor; Then adding mass fraction by stream in reactor is that the pH of 6～10% hydrochloric acid control solution is 6.0, digests hydrolysis, keeps simultaneously 120 ℃ of temperature, pressure 0.1MPa, is hydrolyzed 25 minutes, extracts hydrolyzate; Add weight fraction in the hydrolyzate and be the mixed amino acid solution that valine, L-Trp, tryptophan, sulphur amino acid that 15% corn steep liquor and weight ratio be 1 ︰, 1 ︰, 1 ︰, 1 ︰ 1 form, be made into the organic nitrogen source that solid content is weight fraction 16%～18%, ratio is that the mixing quality ratio of hydrolyzate, corn steep liquor and mixed amino acid solution is 1 ︰, 1 ︰ 1.
5, the preparation of microbial inoculum:
After the fermentation ends, reduce fermentation ventilating ratio to 1 ︰ 0.08, keep fermentation tank pressure 0.02MPa, reduce speed of agitator to 20rpm, zymotic fluid is cooled to below 10 ℃;
By the membrane filtration concentration, fermentating liquid volume is concentrated into original 1/5～1/8.Add 0.07% allotment buffer protection agent (solution of the pH8.0 that sodium dihydrogen phosphate and sodium hydrogen phosphate mix) of concentrated liquid accumulated amount, after mixing, the zymotic fluid after concentrated is carried out spray drying treatment, obtain the microbial inoculum product.
Described spray drying process is: 120 ℃ the hot air sterilization that drying tower, cyclone separator is carried out 30 minutes, then reduce hot blast temperature to 80 ℃, open successively air-introduced machine, fan blower, heater, feed pump, wind stopping device, adjust mass flow to 150～180Kg/h, ventilation to 5000～8000m
3/ h opens membrane pump and pumps into deployed zymotic fluid, carries out the atomized drying of product, obtains the finished product microbial inoculum, and bacterium opaque figureofmerit is: moisture is less than 3.5%, and every gram bacterium number is greater than 2,000 hundred million, and the thalline spore forming rate is greater than 85%;
A large amount of times that occur of gemma are conducive to the formation of gemma all in zymogenic exponential phase and later a period of time in the meta-alkali environment.Four growth period-period of delays, exponential phase, earlier fermentation and fermentation later stage that fermentation process divides; Be about 2～3h period of delay, fermenting entered logarithmic growth period in 9 hours, thalli growth very rapidly, the thalline quantity growth is very fast, fermenting had 10% sporulation in 13 hours.Not regulating in the worth situation of pH, descending in front 11 hours pH values of fermenting always, drop to 6.0 from 7.2; Ferment after 13 hours, bacterial classification begins gradually secreted alkaline albumen antibiotic, and pH begins to rise gradually, finally reaches more than 7.7.In this process, add organic nitrogen source by the later stage, so that thalline forms the metabolic growth, the basic protein antibiotic has improved more than the 30ng/ml, is beneficial to simultaneously the normal generation of gemma, finally obtains a kind of bacillus subtilis agent of Effective Anti tobacco bacterial wilt.
The advantage of comparing the inventive method with existing method is except obtaining forming the dormancy thalline of gemma, also obtained improving the above basic protein antibiotic of 30ng/ml, simultaneously, having improved the thalline spore forming rate and reached more than 85%.
Using method and the usage amount of microbial inoculum of the present invention are as follows:
1, makes the bacterium liquid of the about 2,000 ten thousand/mL of cell concentration, be about a gram bacterium powder and be mixed with 1 liter of bacterium liquid.
2, morbidity Tobacco Root position, field, outer surface are evenly sprayed antibiotic bacterium liquid; Perhaps in the cigarette seedling migration process, section sprays to the cigarette shoot root, the about 2,000 ten thousand/mL of cell concentration, the amount of spraying 5ml/ strain, like this consumption still less, better effects if.
3, about every mu of Tobacco using amount 650 restrains.
Diseases prevention, the result for the treatment of of microbial inoculum of the present invention are as follows:
One, control efficiency
1, cigarette seedling culture transferring root is processed:
(1) culture transferring cigarette shoot root section is processed, after three weeks of growing, it is 0.09% that the cigarette seedling diseases is found to resemble.
(2) do not deal with the cigarette seedling incidence of disease 7.1%.
2, transplant seedlings before, do not do spraying processing, after transplanting seedlings, the field tobacco sprayed to process and spray to process contrast:
(1) field tobacco spray is done executed processing, the tobacco incidence of disease is 0.36%.
(2) do not deal with the cigarette seedling incidence of disease 7.1%.
As can be seen from the above results the incidence of disease that sprays processing not being done in the spray of field tobacco is to do about 20 times that spray processing, and this microbial inoculum control efficiency is good.
Two, result for the treatment of
Morbidity tobacco microbial inoculum sprays processing: to the morbidity diseased plant that occurs, the bacterium liquid that carries out the about 3,000 ten thousand/mL of cell concentration sprays root, the diseased plant surface of morbidity bacterial strain, about the every strain 50ml of the amount of spraying; Blank only sprays water to the morbidity bacterial strain.
The result: for the tobacco leaf that sprays water, after 3 days, 94% is withered after withered rate reached 49%, 6 day.Through the diseased plant that bacterium liquid is processed, after 3 days, 91% diseased plant onset state is tentatively controlled, and 87% morbidity strain obtains taking a turn for the better after 6 days, and subsequently continued growth has shown preferably result for the treatment of.Illustrate that through after the bacterium liquid processing of the present invention better result for the treatment of can be arranged, reduce tobacco grower's loss.
The invention has the advantages that: in this process of thalline fermentation, add organic nitrogen source by the later stage, so that thalline forms the metabolic growth, improve the antibiotic generation of basic protein, be beneficial to simultaneously the normal generation of gemma, finally obtain a kind of bacillus subtilis agent of Effective Anti tobacco bacterial wilt.
Embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, is not intended to limit the present invention.
Except as otherwise noted, the percentage that adopts among the present invention is percetage by weight.
Embodiment 1
A kind of bacillus subtilis microbial agent of anti-tobacco bacterial wilt, its preparation method may further comprise the steps:
1, actication of culture: to the bacillus subtilis B908 activation culture of the slant preservation bought from the market to bacillus exfoliation;
Activation medium (NA) prescription is: beef peptone 3g, and peptone 5g, agar 16g, water is settled to 1000ml;
2, shaking table enlarges cultivation: the bacillus subtilis after will activating is inoculated into and carries out shaking table expansion cultivation in the liquid nutrient medium, and initial pH value is 7.0, and cultivation temperature is 30 ℃, and shaking speed is 200rpm, till 15% bacillus exfoliation;
The prescription of used liquid nutrient medium is: corn flour 6.5g, glucose 2.5g, beancake powder 10.1g, fish meal 2.5g, CaCO
33.5g, (NH
4)
2SO
40.5g, K
2HPO
40.15g, MgSO
47H
2O 0.1g, MnSO
4H
2O 0.1g, water is settled to 500ml;
3, seeding tank enlarges cultivation: under sterile working, shaking table is enlarged the bacterium liquid that obtains after the cultivation be linked in the seeding tank that contains the seed tank culture base, the initial stage ventilates and makes a gesture of measuring 1 ︰ 0.25, later stage ventilating ratio 1 ︰ 1; Simultaneously, the pH value of controlled fermentation liquid is 6.9, and temperature is 32 ℃, and the speed of agitator of fermentation tank is 80rpm; Enter the later stage of exponential phase to bacillus;
Used seed tank culture based formulas is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
4, ferment tank
Change the seeding tank bacterium liquid that is cultured to later stage exponential phase of growth over to fermentation tank that fermentation tank culture medium is housed under aseptic condition, canned amount is 70% of fermentation tank volume, carries out the early stage of fermentation tank and cultivates and the later stage cultivation;
(1) fermentation tank is cultivated in earlier stage: to the fermentation tank of access seed liquor, 28 ℃ of temperature, pH6.9, ventilation 1 ︰ 0.25, speed of agitator 150rpm; Keep zymotic fluid total sugar content 0.35%, total nitrogen content 30ng/ml, be cultured to spore forming rate 55%, enter the fermentation later stage;
(2) the fermentation later stage cultivates: no longer regulate zymotic fluid pH value, use the total nitrogen content of organic nitrogen source after fermentation liquid, control total nitrogen content more than 40ng/ml, be cultured to comprehensive spore forming rate more than 80%, the pH value is more than 7.7, fermentation ends;
The prescription of described fermentation tank culture medium is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
The preparation method of described organic nitrogen source is: will wait the groundnut meal, soybean cake powder, cotton seed powder cake, dusty yeast, dried silkworm chrysalis meal, fish meal of quality to mix, and be modulated into weight fraction and be 9%～16% mixed solution, and join in the reactor; Then adding mass fraction by stream in reactor is that the pH of 6～10% hydrochloric acid control solution is 6.0, digests hydrolysis, keeps simultaneously 120 ℃ of temperature, pressure 0.1MPa, is hydrolyzed 25 minutes, extracts hydrolyzate; Add weight fraction in the hydrolyzate and be 15% corn steep liquor and etc. the mixed amino acid solution that forms of valine, L-Trp, tryptophan, the sulphur amino acid of quality, be made into the organic nitrogen source that solid content is weight fraction 16%～18%, ratio is that the mixing quality ratio of hydrolyzate, corn steep liquor and mixed amino acid solution is 1 ︰, 1 ︰ 1;
5, the preparation of microbial inoculum:
After the fermentation ends, reduce fermentation ventilating ratio to 1 ︰ 0.08, keep fermentation tank malleation 0.02MPa, reduce speed of agitator to 20rpm, zymotic fluid is cooled to below 10 ℃;
By the membrane filtration concentration, fermentating liquid volume is concentrated into original 1/5.After the 0.07% allotment buffer protection agent (being the mixed solution of the pH8.0 that is made into of sodium dihydrogen phosphate and sodium hydrogen phosphate) of adding the concentrated liquid accumulated amount mixes, to the distribution spray drying treatment after concentrating;
Described spray drying process is: 120 ℃ the hot air sterilization that drying tower, cyclone separator is carried out 30 minutes, then reduce hot blast temperature to 80 ℃, open successively air-introduced machine, fan blower, heater, feed pump, wind stopping device, adjust mass flow 150Kg/ hour, ventilation 5000m
3/ hour, open membrane pump and pump into deployed zymotic fluid, carry out the atomized drying of product, obtain the finished product microbial inoculum.
Embodiment 2
A kind of bacillus subtilis microbial agent of anti-tobacco bacterial wilt, its preparation method may further comprise the steps:
1, actication of culture: to the bacillus subtilis B908 activation culture of the slant preservation bought from the market to bacillus exfoliation;
Activation medium (NA) prescription is: beef peptone 3g, and peptone 5g, agar 16g, water is settled to 1000ml;
2, shaking table enlarges cultivation: the bacillus subtilis after will activating is inoculated into and carries out shaking table expansion cultivation in the liquid nutrient medium, and initial pH value is 7.1, and cultivation temperature is 30 ℃, and shaking speed is 200rpm, till 18% bacillus exfoliation;
The prescription of used liquid nutrient medium is: corn flour 6.5g, glucose 2.5g, beancake powder 10.1g, fish meal 2.5g, CaCO
33.5g, (NH
4)
2SO
40.5g, K
2HPO
40.15g, MgSO
47H
2O 0.1g, MnSO
4H
2O 0.1g, water is settled to 500ml;
3, seeding tank enlarges cultivation: under sterile working, the bacterium liquid that shaking table is enlarged after cultivating is linked in the seeding tank that contains the seed tank culture base, and the initial stage ventilates and makes a gesture of measuring 1 ︰ 0.25, later stage ventilating ratio 1 ︰ 1; Simultaneously, the pH value of controlled fermentation liquid is 7.1, and temperature is 32 ℃, and the speed of agitator of fermentation tank is 150 rpm; Enter the later stage of exponential phase to bacillus;
Used seed tank culture based formulas is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
4, ferment tank
Change the seeding tank bacterium liquid that is cultured to later stage exponential phase of growth over to fermentation tank that fermentation tank culture medium is housed under aseptic condition, canned amount is 70% of fermentation tank volume, carries out the early stage of fermentation tank and cultivates and the later stage cultivation;
(1) fermentation tank is cultivated in earlier stage: to the fermentation tank of access seed liquor, 30 ℃ of temperature, pH7.1, ventilation 1 ︰ 0.7, speed of agitator 165 rpm; Keep zymotic fluid total sugar content 0.5%, total nitrogen content 38ng/ml, be cultured to spore forming rate 60%, enter the fermentation later stage;
(2) the fermentation later stage cultivates: no longer regulate zymotic fluid pH value, use the total nitrogen content of organic nitrogen source after fermentation liquid, control total nitrogen content more than 40ng/ml, be cultured to comprehensive spore forming rate more than 80%, the pH value is more than 7.7, fermentation ends;
The prescription of described fermentation tank culture medium is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
The preparation method of described organic nitrogen source is: will wait the groundnut meal, soybean cake powder, cotton seed powder cake, dusty yeast, dried silkworm chrysalis meal, fish meal of quality to mix, and be modulated into weight fraction and be 13% mixed solution, and join in the reactor; Then adding mass fraction by stream in reactor is that the pH of 8% hydrochloric acid control solution is 6.0, digests hydrolysis, keeps simultaneously 120 ℃ of temperature, pressure 0.1MPa, is hydrolyzed 25 minutes, extracts hydrolyzate; Add weight fraction in the hydrolyzate and be 15% corn steep liquor and by etc. the mixed amino acid solution that forms of valine, L-Trp, tryptophan, the sulphur amino acid of quality, be made into the organic nitrogen source that solid content is weight fraction 17%, ratio is that the mixing quality ratio of hydrolyzate, corn steep liquor and mixed amino acid solution is 1 ︰, 1 ︰ 1;
5, the preparation of microbial inoculum:
After the fermentation ends, reduce fermentation ventilating ratio to 1 ︰ 0.08, keep fermentation tank malleation 0.02MPa, reduce speed of agitator to 20rpm, zymotic fluid is cooled to below 10 ℃;
By the membrane filtration concentration, fermentating liquid volume is concentrated into original 1/6.After the 0.07% allotment buffer protection agent (being the mixed solution of the pH8.0 that is made into of sodium dihydrogen phosphate and sodium hydrogen phosphate) of adding the concentrated liquid accumulated amount mixes, to the distribution spray drying treatment after concentrating;
Described spray drying process is: 120 ℃ the hot air sterilization that drying tower, cyclone separator is carried out 30 minutes, then reduce hot blast temperature to 80 ℃, open successively air-introduced machine, fan blower, heater, feed pump, wind stopping device, adjust mass flow 165Kg/ hour, ventilation 6500m
3/ hour, open membrane pump and pump into deployed zymotic fluid, carry out the atomized drying of product, obtain the finished product microbial inoculum.
Embodiment 3
A kind of bacillus subtilis microbial agent of anti-tobacco bacterial wilt, its preparation method may further comprise the steps:
1, actication of culture: to the bacillus subtilis B908 activation culture of the slant preservation bought from the market to bacillus exfoliation;
Activation medium (NA) prescription is: beef peptone 3g, and peptone 5g, agar 16g, water is settled to 1000ml;
2, shaking table enlarges cultivation: the bacillus subtilis after will activating is inoculated into and carries out shaking table expansion cultivation in the liquid nutrient medium, and initial pH value is 7.2, and cultivation temperature is 30 ℃, and shaking speed is 200rpm, till 20% bacillus exfoliation;
The prescription of used liquid nutrient medium is: corn flour 6.5g, glucose 2.5g, beancake powder 10.1g, fish meal 2.5g, CaCO
33.5g, (NH
4)
2SO
40.5g, K
2HPO
40.15g, MgSO
47H
2O 0.1g, MnSO
4H
2O 0.1g, water is settled to 500ml;
3, seeding tank enlarges cultivation: under sterile working, the bacterium liquid that shaking table is enlarged after cultivating is linked in the seeding tank that contains the seed tank culture base, and the initial stage ventilates and makes a gesture of measuring 1 ︰ 0.25, later stage ventilating ratio 1 ︰ 1; Simultaneously, the pH value of controlled fermentation liquid is 7.3, and temperature is 32 ℃, and the speed of agitator of fermentation tank is 220 rpm; Enter the later stage of exponential phase to bacillus;
Used seed tank culture based formulas is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
4, ferment tank
Change the seeding tank bacterium liquid that is cultured to later stage exponential phase of growth over to fermentation tank that fermentation tank culture medium is housed under aseptic condition, canned amount is 70% of fermentation tank volume, carries out the early stage of fermentation tank and cultivates and the later stage cultivation;
(1) fermentation tank is cultivated in earlier stage: to the fermentation tank of access seed liquor, 32 ℃ of temperature, pH7.3, ventilation 1 ︰ 1.1, speed of agitator 180 rpm; Keep zymotic fluid total sugar content 0.6%, total nitrogen content 45ng/ml, be cultured to spore forming rate 65%, enter the fermentation later stage;
(2) the fermentation later stage cultivates: no longer regulate zymotic fluid pH value, use the total nitrogen content of organic nitrogen source after fermentation liquid, control total nitrogen content more than 40ng/ml, be cultured to comprehensive spore forming rate more than 80%, the pH value is more than 7.7, fermentation ends;
The prescription of described fermentation tank culture medium is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
The preparation method of described organic nitrogen source is: will wait the groundnut meal, soybean cake powder, cotton seed powder cake, dusty yeast, dried silkworm chrysalis meal, fish meal of quality to mix, and be modulated into weight fraction and be 16% mixed solution, and join in the reactor; Then adding mass fraction by stream in reactor is that the pH of 6～10% hydrochloric acid control solution is 6.0, digests hydrolysis, keeps simultaneously 120 ℃ of temperature, pressure 0.1MPa, is hydrolyzed 25 minutes, extracts hydrolyzate; Add weight fraction in the hydrolyzate and be 15% corn steep liquor and by etc. the mixed amino acid solution that forms of valine, L-Trp, tryptophan, the sulphur amino acid of quality, be made into the organic nitrogen source that solid content is weight fraction 18%, ratio is that the mixing quality ratio of hydrolyzate, corn steep liquor and mixed amino acid solution is 1 ︰, 1 ︰ 1;
5, the preparation of microbial inoculum:
After the fermentation ends, reduce fermentation ventilating ratio to 1 ︰ 0.08, keep fermentation tank malleation 0.02MPa, reduce speed of agitator to 20rpm, zymotic fluid is cooled to below 10 ℃;
By the membrane filtration concentration, fermentating liquid volume is concentrated into original 1/8.After the 0.07% allotment buffer protection agent (being the mixed solution of the pH8.0 that is made into of sodium dihydrogen phosphate and sodium hydrogen phosphate) of adding the concentrated liquid accumulated amount mixes, to the distribution spray drying treatment after concentrating;
Described spray drying process is: 120 ℃ the hot air sterilization that drying tower, cyclone separator is carried out 30 minutes, then reduce hot blast temperature to 80 ℃, open successively air-introduced machine, fan blower, heater, feed pump, wind stopping device, adjust mass flow 180Kg/ hour, ventilation 8000m
3/ hour, open membrane pump and pump into deployed zymotic fluid, carry out the atomized drying of product, obtain the finished product microbial inoculum.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment the present invention is had been described in detail, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the preparation method of the bacillus subtilis microbial agent of an anti-tobacco bacterial wilt is characterized in that: be prepared from by following methods:
(1) actication of culture: the bacillus subtilis B908 to preservation carries out activation culture to bacillus exfoliation;
(2) shaking table enlarges cultivation: the bacillus subtilis after will activating is inoculated into and carries out shaking table expansion cultivation in the liquid nutrient medium, and initial pH value is 7.0～7.2, and cultivation temperature is 30 ℃, and shaking speed is 200rpm, till 15～20% bacillus exfoliation;
(3) seeding tank enlarges cultivation: under sterile working, the bacterium liquid that obtains is cultivated in shaking table expansion be linked in the seeding tank that contains the seed tank culture base initial stage ventilating ratio 1 ︰ 0.25, later stage ventilating ratio 1 ︰ 1; Simultaneously, the pH value of controlled fermentation liquid is 6.9～7.3, and temperature is 32 ℃, and the speed of agitator of fermentation tank is 80～220 rpm; Enter the later stage of exponential phase to bacillus;
(4) ferment tank: the seeding tank bacterium liquid that will be cultured to later stage exponential phase of growth changes the fermentation tank that fermentation tank culture medium is housed under aseptic condition, canned amount is 70% of fermentation tank volume, carries out the early stage of fermentation tank and cultivates and the later stage cultivation;
(5) preparation of microbial inoculum: after ferment tank finishes, reduce fermentation ventilating ratio to 1 ︰ 0.08, keep fermentation tank pressure 0.02MPa, reduce speed of agitator to 20rpm, zymotic fluid is cooled to below 10 ℃; Then by the membrane filtration concentration, fermentating liquid volume is concentrated into original 1/5～1/8; Add again the agent of allotment buffer protection, after mixing, the zymotic fluid after concentrated is carried out spray drying treatment, obtain the microbial inoculum product;
The used culture medium prescription of described step (1) activation is: beef peptone 3g, and peptone 5g, agar 16g, water is settled to 1000ml; The prescription of the liquid nutrient medium of described step (2) is: corn flour 6.5g, glucose 2.5g, beancake powder 10.1g, fish meal 2.5g, CaCO
33.5g, (NH
4)
2SO
40.5g, K
2HPO
40.15g, MgSO
47H
2O 0.1g, MnSO
4H
2O 0.1g, water is settled to 500ml;
The seed tank culture based formulas of described step (3) is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
The prescription of the fermentation tank culture medium of described step (4) is: corn flour 12.98g, glucose 5g, beancake powder 20.1g, fish meal 5g, CaCO
36.90g, (NH
4)
2SO
41g, K
2HPO
40.3g, MgSO
47H
2O 0.2g, MnSO
4H
2O 0.2g, water is settled to 1000ml;
The fermentation tank cultural method in early stage of described step (4) is: to the fermentation tank of access seed liquor, 28～32 ℃ of temperature, pH6.9～7.3, ventilation 1 ︰ 0.25～1.1, speed of agitator 150～180 rpm; Keep zymotic fluid total sugar content 0.35～0.6%, total nitrogen content 30～45ng/ml, be cultured to spore forming rate 55～65%, enter the fermentation later stage;
The fermentation tank later stage cultural method of described step (4) is: use organic nitrogen source to regulate the total nitrogen content of zymotic fluid, the control total nitrogen content is cultured to spore forming rate 80% or more, pH value more than 7.7, the later stage fermentation end more than 40ng/ml.
2. the preparation method of the bacillus subtilis microbial agent of a kind of anti-tobacco bacterial wilt according to claim 1, it is characterized in that: the preparation method of the organic nitrogen source of described step (4) is: be that the ratio of 1 ︰, 1 ︰, 1 ︰, 1 ︰, 1 ︰ 1 is mixed with groundnut meal, soybean cake powder, cotton seed powder cake, dusty yeast, dried silkworm chrysalis meal, fish meal according to weight ratio, be modulated into weight fraction and be 9%～16% mixed solution, join in the reactor; Then adding mass fraction by stream in reactor is that the pH of 6～10% hydrochloric acid control solution is 6.0, digests hydrolysis, keeps simultaneously 120 ℃ of temperature, pressure 0.1MPa, is hydrolyzed 25 minutes, extracts hydrolyzate; Add weight fraction in the hydrolyzate and be the mixed amino acid solution that valine, L-Trp, tryptophan, sulphur amino acid that 15% corn steep liquor and weight ratio be 1 ︰, 1 ︰, 1 ︰, 1 ︰ 1 form, be made into the organic nitrogen source that solid content is weight fraction 16%～18%, ratio is that the mixing quality ratio of hydrolyzate, corn steep liquor and mixed amino acid solution is 1 ︰, 1 ︰ 1.
3. the preparation method of the bacillus subtilis microbial agent of a kind of anti-tobacco bacterial wilt according to claim 1, it is characterized in that: the spray drying process of described step (5) is: 120 ℃ the hot air sterilization that drying tower, cyclone separator is carried out 30 minutes, then reduce hot blast temperature to 80 ℃, open successively air-introduced machine, fan blower, heater, feed pump, wind stopping device, adjust mass flow to 150～180Kg/h, ventilation to 5000～8000m
3/ h opens membrane pump and pumps into deployed zymotic fluid, carries out the atomized drying of product, obtains the finished product microbial inoculum, and bacterium opaque figureofmerit is: moisture is less than 3.5%, and every gram bacterium number is greater than 2,000 hundred million, and the thalline spore forming rate is greater than 85%.
4. the preparation method of the bacillus subtilis microbial agent of a kind of anti-tobacco bacterial wilt according to claim 1; it is characterized in that: the agent of described allotment buffer protection is the solution of the pH8.0 that mixed by sodium dihydrogen phosphate and sodium hydrogen phosphate, and addition is 0.07% of concentrated liquid accumulated amount.
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| CN106007967A (en) * | 2016-05-19 | 2016-10-12 | 山东农保姆肥业科技有限公司 | Preparation method of microbe leaf fertilizer |
| CN106495961A (en) * | 2016-12-21 | 2017-03-15 | 潍坊市信得生物科技有限公司 | The strong plantlets and rootage fertilizer that a kind of liquid fermentation production makes |
| CN108849985B (en) * | 2018-05-30 | 2021-02-26 | 唐山市农业科学研究院 | Ternary compound biological agent and application thereof in prevention and treatment of ginger bacterial wilt |
| CN109182214A (en) * | 2018-10-17 | 2019-01-11 | 湖北省烟草科学研究院 | Synergy biocontrol microorganisms and its preparation, application method for tobacco bacterial wilt prevention and treatment |
| CN109468259B (en) * | 2018-10-24 | 2022-04-05 | 亚太星原农牧科技海安有限公司 | Culture medium for promoting spore generation |
| CN112586509A (en) * | 2020-11-13 | 2021-04-02 | 遵义医科大学 | Compound microorganism effervescent tablet for preventing tobacco bacterial wilt and promoting growth, and preparation method and application thereof |
| CN112961012A (en) * | 2021-03-09 | 2021-06-15 | 石河子市郁茏生物科技有限公司 | Preparation method of microbial drip irrigation fertilizer |
| CN114672428A (en) * | 2021-12-30 | 2022-06-28 | 云南省微生物发酵工程研究中心有限公司 | Biological preparation for preventing and treating tobacco bacterial wilt, preparation method and application |
| CN115029276B (en) * | 2022-06-24 | 2023-07-04 | 中国烟草总公司四川省公司 | Acid-resistant antagonistic bacterium of bacterial wilt of tobacco and application thereof |
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