CN105886430A - Method for producing bacillus amyloliquefaciens bacterial agent through solid-state fermentation - Google Patents
Method for producing bacillus amyloliquefaciens bacterial agent through solid-state fermentation Download PDFInfo
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Abstract
The invention relates to a method for producing a bacillus amyloliquefaciens bacterial agent through solid-state fermentation. Wheat bran, soybean meal and corn flour are used as main raw materials to make a solid-state fermentation culture medium. The made culture medium is wetted with water, standing culture is performed at the temperature of 28-30 DEG C for 7-8 hours, and then the culture medium is mixed with a chlorine dioxide disinfectant for sterilization at room temperature; the culture medium subjected to sterilization is inoculated with a bacillus amyloliquefaciens seed solution by using the chlorine dioxide disinfectant for solid-state fermentation; after the fermentation is completed, and a solid-state fermented mash is subjected to hot-air drying and smashing to obtain the bacillus amyloliquefaciens bacterial agent, wherein the spore number of the fungicide is up to 200-22 billion/g, and the spore rate is up to 97%-99%. The method adopts chlorine dioxide sterilization to replace traditional high-temperature steam sterilization and is short in time and high in efficiency, the production costs of the bacillus amyloliquefaciens bacterial agent are greatly reduced, and environmental pollution is reduced.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of solid fermentation and produce bacillus amyloliquefaciens bacterium
The method of agent.
Background technology
Agricultural production often has corps diseases occur, cause great economic loss to people.For
Preventing and treating corps diseases, generally uses chemical pesticide.Chemical pesticide has low cost, instant effect, saves time
The advantage such as laborsaving, but use chemical pesticide in a large number, not only environment is caused pollution greatly, and makes agriculture
Deterioration in quality, poisonous and harmful substance exceeds standard even to cause agriculture residual etc..Biological pesticide refers to utilize biological living
Body or its metabolite carry out the preparation killed or suppress for agricultural pest, have safe efficient,
The feature such as pollution-free.Along with people and government's environmental consciousness and the enhancing of health care consciousness, the biological agriculture of research and development
Medicine replaces chemical pesticide to become the inexorable trend of social development.
Microbial pesticide is the class being most widely used in biological pesticide.The main component of microbial pesticide
It is living microorganisms and secondary metabolite such as antibiotic etc. thereof.With the biological group of used as pesticides mainly have antibacterial,
Fungus and virus etc..Microbial pesticide has parasite killing, sterilization, weeding and plant growth regulation.From
So for biocontrol bacteria in boundary, mainly there are bacillus, Rhodopseudomonas.Microbial pesticide is considered
Being public nuisance-free agricultural chemicals, controlling object is not likely to produce Drug resistance, does not injure natural enemy, and breeding is fast, can utilize agricultural and sideline product
Product even industrial and agricultural wastewater, garbage etc. produce.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) belongs to bacillus (Bacillus)
Gram-positive bacterium, be a kind of bacillus mesophilic, aerobic, sporiferous.This bacterium is divided at nature
Cloth is extensive, nontoxic to people and animals, free from environmental pollution, and fast growth can produce multiple antibacterial substance,
There is suppression antibacterial and the fungi activity of wide spectrum, to the root rot of plant, droop, anthrachose of grape green mould etc.
There is fine preventive and therapeutic effect.Bacillus amyloliquefaciens has been used for producing microbial pesticide, agricultural microorganism bacterium
Agent and biological organic fertilizer.
At present bacillus amyloliquefaciens uses liquid submerged fermentation technology, more spray-dried produces, right
Equipment requirements is high, complex manufacturing.Comparing liquid submerged fermentation, solid fermentation has the advantage that training
Support based raw material wide material sources and price is low；The equipment used is relatively easy, and energy consumption is low, and production cost is far below
Liquid submerged fermentation；Sweat not waste discharge, environmental pollution is less.The more important thing is, use solid-state
Antibiotic substance produced by thalline can be preserved by fermentation completely, and uses liquid submerged fermentation easily to make these
Antibiotic substance is discharged with waste water.
Disinfection agent of chlorine dioxide is a kind of strong oxidizer, has high-efficiency broad spectrum sterilization or bacteriostasis, Er Qiean
The most free from environmental pollution, the high effect disinfectants of A1 level it is classified as by World Health Organization (WHO).Chlorine dioxide to antibacterial,
Fungus, virus and parasite etc. are respectively provided with the good effect killed or suppress, and degradable, noresidue,
At home and abroad have been widely used for drinking water treatment, food fresh keeping anticorrosion, waste water processes, indoor environment disappears
The fields such as poison.Although chlorine dioxide also has stronger suppression make the spore of various bacillus, the spore of mycete
With, but spore and spore can not be killed completely.
Bacillus amyloliquefaciens solid state fermentation culture method is less.Such as, Chinese patent (publication number: CN
Solid-state fermentation culture medium in 104830711A) (45% soy sauce bean cake, 15% Testa oryzae, 0.12% magnesium sulfate,
0.12% manganese sulfate) 115～128 DEG C of high temperature sterilizes 20～35min, inoculate bacillus amyloliquefaciens kind after cooling
Sub-liquid, 24-36h cultivated by 37-42 DEG C of disk.With flower in Chinese patent (publication number: CN 104962492A)
Raw cake powder, Testa Tritici, cottonseed meal are that primary raw material composition states solid medium.Solid medium 120～125 DEG C go out
Bacterium twice, each sterilizing 30～45min.Exist solving the starch spore bar seed liquor mixture with solid medium
Quiescent culture 5d under the conditions of 30 DEG C.
The weak point that above patented technology method exists is:
1. high temperature sterilization needs to consume substantial amounts of heat energy.These heat energy are either provided by coal burning, or
Thered is provided by electric energy, all can be greatly increased production cost, be caused directly or indirectly environmental pollution.
2. high temperature sterilize and the cooling of solid medium are time-consuming long, and production efficiency is relatively low.
3. high temperature sterilize can cause the destruction of solid medium Middle nutrition material, does not utilize thalli growth.
Summary of the invention
The solid fermentation that it is an object of the invention to provide a kind of low cost produces bacillus amyloliquefaciens method.Solve
In the solid ferment process of bacillus amyloliquefaciens, disinfection agent of chlorine dioxide is used to replace traditional high temperature sterilization
Kill the miscellaneous bacterias such as the antibacterial in culture medium, yeast, mycete, thus greatly reduce production cost.Given birth to
Produce bacillus amyloliquefaciens microbial inoculum, its spore number reaches 200～22,000,000,000/g, spore rate reach 97～
99%.
The bacterial strain that invention is used is a plant height effect antagonism various plants pathogen, promotes the Xie Dian of plant growing
Afnyloliquefaciens ((Bacillus amyloliquefaciens) B3.
The principle of the present invention: disinfection agent of chlorine dioxide can kill the thin of the microorganisms such as antibacterial, yeast, mycete
Born of the same parents or mycelia, but the spore of antibacterial and the spore of mycete can not be killed.Solid medium is added a small amount of water system
Become the water-containing medium of bean curd scoriform, cultivate a period of time at 28～30 DEG C, its objective is to allow in culture medium
Bacterial spore and mycotic spore sprout；Then with disinfection agent of chlorine dioxide, this culture medium is carried out disinfection, kill
The microorganisms such as dead antibacterial therein, yeast, mycete, reach culture medium the most aseptic.Solid-state training after sterilization
Supporting in base, inoculation bacillus amyloliquefaciens seed liquor carries out solid fermentation.Bacillus amyloliquefaciens itself can produce
Raw antibiotic substance such as antibacterial peptide, antibacterial protein etc., have a stronger antibacterial ability, and fast growth, because of
This, even if remaining miscellaneous bacteria after Xiao Du in solid medium, these miscellaneous bacterias can not grow, thus ensures whole
Being smoothed out of individual sweat.After fermentation ends, solid fermentation wine with dregs is carried out hot air drying, pulverizing, i.e.
Obtain bacillus amyloliquefaciens microbial inoculum.
The present invention is achieved by the following technical solutions:
A kind of solid fermentation produces the method for bacillus amyloliquefaciens microbial inoculum, comprises the following steps:
A, the preparation of seed culture medium and sterilizing: in 1L water, add 20～30g glucoses, 10～20g
Peptone, 5～10g yeast powder, 2～2.5 ammonium sulfate, 5.0～5.5g sodium chloride, 4.0～4.5g phosphoric acid
Potassium dihydrogen, 1.8～2.0g calcium carbonate, according to described proportioning, add after being weighed by various compositions in triangular flask,
Stir under room temperature, prepare seed culture medium；Put in high-pressure sterilizing pot after sealing with 8 layers of gauze,
115 DEG C～121 DEG C of sterilizings 15～20min, be cooled to room temperature triangular flask after sterilizing；
B, the preparation of seed liquor: bacillus amyloliquefaciens B3 strain 2～3 ring of picking slant preservation, inoculation
In the seed culture medium described in step a, 30 DEG C, 180～200rpm shaking table shaken cultivation 12～14h；
C, the pretreatment of solid-state fermentation culture medium: 100g solid-state fermentation culture medium by 70～80g Testa Tritici,
15～20g bean cake powders, 5～10g Semen Maydis powder composition；According to above-mentioned weight proportion, by Testa Tritici, bean
Dregs of rice powder, Semen Maydis powder mix homogeneously, be solid-state fermentation culture medium；In 1000g solid-state fermentation culture medium,
Add 200～250mL tap waters, mix homogeneously so that it is in moisture state, be positioned in incubator 28 DEG C～
30 DEG C of quiescent culture 7～8h；
D, the preparation of chlorine dioxide disinfection liquid: by 2～3 disinfection by chlorine dioxide effervescent tablets (1g/ sheet, dioxies
Change chlorinity 12%) join in 1L water, treat that effervescent tablet is completely dissolved, and adjust its pH with 0.2M hydrochloric acid
Value is 6.0～6.2, and i.e. making concentration is 240～360mg/L chlorine dioxide disinfection liquids；
E, the sterilization of solid-state fermentation culture medium: in the 1000g solid-state fermentation culture medium described in step c, add
Enter 4～5g potassium dihydrogen phosphates, potassium chloride 2.5～3g, sodium chloride 5.0～6g, transfer to carry organic after mixing
In the horizontal rustless steel solid-state fermenter of tool stirring paddle, add the dioxy described in 350～400mL steps d
Change chlorination liquid, stir under room temperature, stand sterilization 15～20min；
F, solid fermentation: start the solid-state fermentation culture medium 8～10min of mechanical agitation oar whipping step e, its
Purpose is to be discharged by the chlorine dioxide of remaining in culture medium, in order to avoid damage solves starch in follow-up cultivation
Bacillus cell, then, inoculates bacillus amyloliquefaciens B3 described in step b with 4%～5% inoculum concentration
In solid-state fermentation culture medium after stirring, control fermentation temperature 30～34 DEG C, every 8h stirs once, often
Secondary stirring 8～10min, fermentation 96～100h, obtain solid fermentation wine with dregs.
G, it is dried: use 60 DEG C～the solid fermentation wine with dregs described in step f is dried water content 30% by 70 DEG C of hot blasts
Below.
H, pulverizing: use beater grinder the dried solid fermentation wine with dregs described in step g is ground into 60～
The powder of 70 mesh, i.e. obtains bacillus amyloliquefaciens B3 microbial inoculum, is packed by bacillus amyloliquefaciens B3 microbial inoculum
After, leave shady and cool being dried in and locate.
Preferably, in described step c 100g solid-state fermentation culture medium by 70g Testa Tritici, 20g bean cake powder,
10g Semen Maydis powder forms.
Preferably, 1000g solid-state fermentation culture medium in described step c, add 200～250mL tap waters,
Mix homogeneously so that it is in moisture state, is positioned over 30 DEG C of quiescent culture 7～8h.
Preferably, in described step d the pH value of chlorine monoxid disinfectant solution be 6.0～6.2, chlorine dioxide concentration
It is 300～360mg/L.
Preferably, in described step e, 1000g solid-state fermentation culture medium adds 380～400mL chlorine dioxide
Disinfectant solution, stirs under room temperature, stands sterilization 18～20min.
Preferably, the spore number of the bacillus amyloliquefaciens microbial inoculum obtained in described step h reach 200～
22000000000/g, spore rate reaches 97%～99%.
Preferably, a, the preparation of seed culture medium and sterilizing: in 1L water, add 30g glucose, 20g
Peptone, 5g yeast powder, 2 ammonium sulfate, 5.0g sodium chloride, 4.0 potassium dihydrogen phosphates, 2.0g calcium carbonate.
According to described proportioning, add in triangular flask after various compositions are weighed, stir under room temperature, prepare seed
Culture medium；Putting in high-pressure sterilizing pot after sealing with 8 layers of gauze, 115 DEG C of sterilizing 20min, three after sterilizing
Angle bottle is cooled to room temperature；
B, the preparation of seed liquor: bacillus amyloliquefaciens B3 strain 2～3 ring of picking slant preservation, inoculation
In the seed culture medium described in step a, 30 DEG C, 200rpm shaking table shaken cultivation 12h；
C, the pretreatment of solid-state fermentation culture medium: 100g solid-state fermentation culture medium is by 70g Testa Tritici, 20g
Bean cake powder, 10g Semen Maydis powder form.According to said ratio, by Testa Tritici, bean cake powder, Semen Maydis powder mixing
Uniformly, it is solid-state fermentation culture medium.In 1000g solid-state fermentation culture medium, add 250mL tap water,
Mix homogeneously so that it is in moisture state, is positioned over quiescent culture 7h in 30 DEG C of incubators；
D, the preparation of chlorine dioxide disinfection liquid: by 3 disinfection by chlorine dioxide effervescent tablets (1g/ sheet, titanium dioxide
Chlorinity 12%) join in 1L water, treat that effervescent tablet is completely dissolved, and adjust its pH value with 0.2M hydrochloric acid
Being 6.0～6.2, i.e. making concentration is 360mg/L chlorine dioxide disinfection liquid；
E, the sterilization of solid-state fermentation culture medium: in the 1000g solid-state fermentation culture medium described in step c, add
Enter 4.0g potassium dihydrogen phosphate, potassium chloride 2.5g, sodium chloride 5.0g, transfer to after mixing with mechanical agitation oar
Horizontal rustless steel solid-state fermenter in, add the chlorine dioxide disinfection liquid described in 400mL step d, room
Stir under temperature, stand sterilization 15min；
F, solid fermentation: start the solid-state fermentation culture medium 8～10min of mechanical agitation oar whipping step e, its
Purpose is to be discharged by the chlorine dioxide of remaining in culture medium, in order to avoid damage solves starch in follow-up cultivation
Bacillus cell.Then, bacillus amyloliquefaciens B3 described in step b is inoculated into 5% inoculum concentration stirs
In solid-state fermentation culture medium after mixing, controlling fermentation temperature 34 DEG C, every 8h stirs once, every time stirring 8～
10min, ferment 96h, obtains solid fermentation wine with dregs；
G, it is dried: use 60 DEG C～the solid fermentation wine with dregs described in step f is dried water content 30% by 70 DEG C of hot blasts
Below；
H, pulverizing: use beater grinder the dried solid fermentation wine with dregs described in step g is ground into 60～
The powder of 70 mesh, i.e. obtains bacillus amyloliquefaciens B3 microbial inoculum, and its spore number reaches 22,000,000,000/g, bud
Spore rate reaches 99%.After microbial inoculum packaging, leave shady and cool being dried in and locate.
The present invention compared with prior art has the beneficial effect that
1. disinfection agent of chlorine dioxide is used to replace traditional high temperature sterilization to kill the antibacterial in culture medium, ferment
The miscellaneous bacterias such as mother, mycete, considerably reduce thermal energy consumption, reduce production cost low, and not
Environment pollution can be caused.
2. solid medium uses disinfection agent of chlorine dioxide to sterilize, and the time is short, and production efficiency is high.
3. disinfection agent of chlorine dioxide carries out the sterilization of solid medium, does not results in the destruction of nutrient substance.
4. disinfection agent of chlorine dioxide consumption is extremely low, and 1000g solid-state fermentation culture medium only needs 84～144mg dioxies
Change chlorine.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment one:
A, the preparation of seed culture medium and sterilizing: in 1L water, add 30g glucose, 20g peptone,
5g yeast powder, 2 ammonium sulfate, 5.0g sodium chloride, 4.0 potassium dihydrogen phosphates, 2.0g calcium carbonate.According to described
Proportioning, adds in triangular flask after being weighed by various compositions, stirs under room temperature, prepares seed culture medium；
Put in high-pressure sterilizing pot after sealing with 8 layers of gauze, 115 DEG C of sterilizing 20min, after sterilizing, triangular flask is cooled down
To room temperature；
B, the preparation of seed liquor: bacillus amyloliquefaciens B3 strain 2～3 ring of picking slant preservation, inoculation
In the seed culture medium described in step a, 30 DEG C, 200rpm shaking table shaken cultivation 12h；
C, the pretreatment of solid-state fermentation culture medium: 100g solid-state fermentation culture medium is by 70g Testa Tritici, 20g
Bean cake powder, 10g Semen Maydis powder form.According to said ratio, by Testa Tritici, bean cake powder, Semen Maydis powder mixing
Uniformly, it is solid-state fermentation culture medium.In 1000g solid-state fermentation culture medium, add 250mL tap water,
Mix homogeneously so that it is in moisture state, is positioned over quiescent culture 7h in 30 DEG C of incubators；
D, the preparation of chlorine dioxide disinfection liquid: by 3 disinfection by chlorine dioxide effervescent tablets (1g/ sheet, titanium dioxide
Chlorinity 12%) join in 1L water, treat that effervescent tablet is completely dissolved, and adjust its pH value with 0.2M hydrochloric acid
Being 6.0～6.2, i.e. making concentration is 360mg/L chlorine dioxide disinfection liquid；
E, the sterilization of solid-state fermentation culture medium: in the 1000g solid-state fermentation culture medium described in step c, add
Enter 4.0g potassium dihydrogen phosphate, potassium chloride 2.5g, sodium chloride 5.0g, transfer to after mixing with mechanical agitation oar
Horizontal rustless steel solid-state fermenter in, add the chlorine dioxide disinfection liquid described in 400mL step d, room
Stir under temperature, stand sterilization 15min；
F, solid fermentation: start the solid-state fermentation culture medium 8～10min of mechanical agitation oar whipping step e, its
Purpose is to be discharged by the chlorine dioxide of remaining in culture medium, in order to avoid damage solves starch in follow-up cultivation
Bacillus cell.Then, bacillus amyloliquefaciens B3 described in step b is inoculated into 5% inoculum concentration stirs
In solid-state fermentation culture medium after mixing, controlling fermentation temperature 34 DEG C, every 8h stirs once, every time stirring 8～
10min, ferment 96h, obtains solid fermentation wine with dregs.
G, it is dried: use 60 DEG C～the solid fermentation wine with dregs described in step f is dried water content 30% by 70 DEG C of hot blasts
Below.
H, pulverizing: use beater grinder the dried solid fermentation wine with dregs described in step g is ground into 60～
The powder of 70 mesh, i.e. obtains bacillus amyloliquefaciens B3 microbial inoculum, and its spore number reaches 22,000,000,000/g, bud
Spore rate reaches 99%.After microbial inoculum packaging, leave shady and cool being dried in and locate.
Embodiment two
A, the preparation of seed culture medium and sterilizing: in 1L water, add 20g glucose, 20g peptone,
10g yeast powder, 2.5 ammonium sulfate, 5.5g sodium chloride, 4.5g potassium dihydrogen phosphate, 1.8g calcium carbonate.According to
Described proportioning, adds in triangular flask after being weighed by various compositions, stirs under room temperature, prepares seed culture
Base；Putting in high-pressure sterilizing pot after sealing with 8 layers of gauze, 121 DEG C of sterilizing 15min, triangular flask after sterilizing
It is cooled to room temperature；
B, the preparation of seed liquor: bacillus amyloliquefaciens B3 strain 2～3 ring of picking slant preservation, inoculation
In the seed culture medium described in step a, 30 DEG C, 180rpm shaking table shaken cultivation 14h；
C, the pretreatment of solid-state fermentation culture medium: 100g solid-state fermentation culture medium is by 80g Testa Tritici, 15g
Bean cake powder, 5g Semen Maydis powder form.According to said ratio, by Testa Tritici, bean cake powder, Semen Maydis powder mixing
Uniformly, it is solid-state fermentation culture medium.In 1000g solid-state fermentation culture medium, add 200mL tap water,
Mix homogeneously so that it is in moisture state, is positioned over 28 DEG C of quiescent culture 8h in incubator；
D, the preparation of chlorine dioxide disinfection liquid: by 2 disinfection by chlorine dioxide effervescent tablets (1g/ sheet, titanium dioxide
Chlorinity 12%) join in 1L water, treat that effervescent tablet is completely dissolved, and adjust its pH value with 0.2M hydrochloric acid
Being 6.0～6.2, i.e. making concentration is 240mg/L chlorine dioxide disinfection liquid；
E, the sterilization of solid-state fermentation culture medium: in the 1000g solid-state fermentation culture medium described in step c, add
Enter 5.0g potassium dihydrogen phosphate, potassium chloride 3.0g, sodium chloride 6.0g, transfer to mechanical agitation after mixing
In the horizontal rustless steel solid-state fermenter of oar, add the chlorine dioxide disinfection liquid described in 350mL step d,
Stir under room temperature, stand sterilization 20min；
F, solid fermentation: start the solid-state fermentation culture medium 8～10min of mechanical agitation oar whipping step e, its
Purpose is to be discharged by the chlorine dioxide of remaining in culture medium, in order to avoid damage solves starch in follow-up cultivation
Bacillus cell.Then, by bacillus amyloliquefaciens B3 described in step b with 4% inoculum concentration inoculation stirring
After solid-state fermentation culture medium in, control fermentation temperature 30 DEG C, every 8h stirs once, every time stirring 8～
10min, ferment 100h, obtains solid fermentation wine with dregs.
G, it is dried: use 60 DEG C～the solid fermentation wine with dregs described in step f is dried water content 30% by 70 DEG C of hot blasts
Below.
H, pulverizing: use beater grinder the dried solid fermentation wine with dregs described in step g is ground into 60～
The powder of 70 mesh, i.e. obtains bacillus amyloliquefaciens B3 microbial inoculum, and its spore number reaches 20,000,000,000/g, bud
Spore rate reaches 97%.After microbial inoculum packaging, leave shady and cool being dried in and locate.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all the present invention's
Within scope of data and principle, any amendment of being made, equal replacement, improvement etc., should be included in this
Within bright protection domain.
Claims (7)
1. the method that solid fermentation produces bacillus amyloliquefaciens microbial inoculum, is characterized in that comprising the following steps:
A, the preparation of seed culture medium and sterilizing: in 1L water, add 20～30g glucoses, 10～20g
Peptone, 5～10g yeast powder, 2～2.5 ammonium sulfate, 5.0～5.5g sodium chloride, 4.0～4.5g phosphoric acid
Potassium dihydrogen, 1.8～2.0g calcium carbonate, according to described proportioning, add after being weighed by various compositions in triangular flask,
Stir under room temperature, prepare seed culture medium；Put in high-pressure sterilizing pot after sealing with 8 layers of gauze,
115 DEG C～121 DEG C of sterilizings 15～20min, be cooled to room temperature triangular flask after sterilizing；
B, the preparation of seed liquor: bacillus amyloliquefaciens B3 strain 2～3 ring of picking slant preservation, inoculation
In the seed culture medium described in step a, 30 DEG C, 180～200rpm shaking table shaken cultivation 12～14h；
C, the pretreatment of solid-state fermentation culture medium: 100g solid-state fermentation culture medium by 70～80g Testa Tritici,
15～20g bean cake powders, 5～10g Semen Maydis powder composition；According to above-mentioned weight proportion, by Testa Tritici, bean
Dregs of rice powder, Semen Maydis powder mix homogeneously, be solid-state fermentation culture medium；In 1000g solid-state fermentation culture medium,
Add 200～250mL tap waters, mix homogeneously so that it is in moisture state, be positioned in incubator 28 DEG C～
30 DEG C of quiescent culture 7～8h；
D, the preparation of chlorine dioxide disinfection liquid: 2～3 disinfection by chlorine dioxide effervescent tablets are joined in 1L water,
Treating that effervescent tablet is completely dissolved, and to adjust its pH value with 0.2M hydrochloric acid be 6.0～6.2, i.e. making concentration is
240～360mg/L chlorine dioxide disinfection liquids；
E, the sterilization of solid-state fermentation culture medium: in the 1000g solid-state fermentation culture medium described in step c, add
Enter 4～5g potassium dihydrogen phosphates, potassium chloride 2.5～3g, sodium chloride 5.0～6g, transfer to carry organic after mixing
In the horizontal rustless steel solid-state fermenter of tool stirring paddle, add the dioxy described in 350～400mL steps d
Change chlorination liquid, stir under room temperature, stand sterilization 15～20min；
F, solid fermentation: start the solid-state fermentation culture medium 8～10min of mechanical agitation oar whipping step e, its
Purpose is to be discharged by the chlorine dioxide of remaining in culture medium, in order to avoid damage solves starch in follow-up cultivation
Bacillus cell, then, inoculates bacillus amyloliquefaciens B3 described in step b with 4%～5% inoculum concentration
In solid-state fermentation culture medium after stirring, control fermentation temperature 30～34 DEG C, every 8h stirs once, often
Secondary stirring 8～10min, fermentation 96～100h, obtain solid fermentation wine with dregs.
G, it is dried: use 60 DEG C～the solid fermentation wine with dregs described in step f is dried water content 30% by 70 DEG C of hot blasts
Below.
H, pulverizing: use beater grinder the dried solid fermentation wine with dregs described in step g is ground into 60～
The powder of 70 mesh, i.e. obtains bacillus amyloliquefaciens B3 microbial inoculum, is packed by bacillus amyloliquefaciens B3 microbial inoculum
After, leave shady and cool being dried in and locate.
Solid fermentation the most according to claim 1 produces the method for bacillus amyloliquefaciens microbial inoculum, and it is special
Levy and be: in described step c, 100g solid-state fermentation culture medium is by 70g Testa Tritici, 20g bean cake powder, 10g
Semen Maydis powder forms.
Solid fermentation the most according to claim 1 produces the method for bacillus amyloliquefaciens microbial inoculum, and it is special
Levy and be: 1000g solid-state fermentation culture medium in described step c, add 200～250mL tap waters, mixing
Uniformly so that it is in moisture state, 30 DEG C of quiescent culture 7～8h it are positioned over.
Solid fermentation the most according to claim 1 produces the method for bacillus amyloliquefaciens microbial inoculum, and it is special
Levy and be: in described step d the pH value of chlorine monoxid disinfectant solution be 6.0～6.2, chlorine dioxide concentration be 300～
360mg/L。
Solid fermentation the most according to claim 1 produces the method for bacillus amyloliquefaciens microbial inoculum, and it is special
Levy and be: in described step e, 1000g solid-state fermentation culture medium adds 380～400mL chlorine dioxide disinfection liquids,
Stir under room temperature, stand sterilization 18～20min.
Solid fermentation the most according to claim 1 produces the method for bacillus amyloliquefaciens microbial inoculum, and it is special
Levying and be, the spore number of the bacillus amyloliquefaciens microbial inoculum obtained in described step h reaches 200～22,000,000,000
/ g, spore rate reaches 97%～99%.
Solid fermentation the most according to claim 1 produces the method for bacillus amyloliquefaciens microbial inoculum, and it is special
Levy is to include step in detail below:
A, the preparation of seed culture medium and sterilizing: in 1L water, add 30g glucose, 20g peptone,
5g yeast powder, 2 ammonium sulfate, 5.0g sodium chloride, 4.0 potassium dihydrogen phosphates, 2.0g calcium carbonate.According to described
Proportioning, adds in triangular flask after being weighed by various compositions, stirs under room temperature, prepares seed culture medium；
Put in high-pressure sterilizing pot after sealing with 8 layers of gauze, 115 DEG C of sterilizing 20min, after sterilizing, triangular flask is cooled down
To room temperature；
B, the preparation of seed liquor: bacillus amyloliquefaciens B3 strain 2～3 ring of picking slant preservation, inoculation
In the seed culture medium described in step a, 30 DEG C, 200rpm shaking table shaken cultivation 12h；
C, the pretreatment of solid-state fermentation culture medium: 100g solid-state fermentation culture medium is by 70g Testa Tritici, 20g
Bean cake powder, 10g Semen Maydis powder form.According to said ratio, by Testa Tritici, bean cake powder, Semen Maydis powder mixing
Uniformly, it is solid-state fermentation culture medium.In 1000g solid-state fermentation culture medium, add 250mL tap water,
Mix homogeneously so that it is in moisture state, is positioned over quiescent culture 7h in 30 DEG C of incubators；
D, the preparation of chlorine dioxide disinfection liquid: by 3 disinfection by chlorine dioxide effervescent tablets (1g/ sheet, titanium dioxide
Chlorinity 12%) join in 1L water, treat that effervescent tablet is completely dissolved, and adjust its pH value with 0.2M hydrochloric acid
Being 6.0～6.2, i.e. making concentration is 360mg/L chlorine dioxide disinfection liquid；
E, the sterilization of solid-state fermentation culture medium: in the 1000g solid-state fermentation culture medium described in step c, add
Enter 4.0g potassium dihydrogen phosphate, potassium chloride 2.5g, sodium chloride 5.0g, transfer to after mixing with mechanical agitation oar
Horizontal rustless steel solid-state fermenter in, add the chlorine dioxide disinfection liquid described in 400mL step d, room
Stir under temperature, stand sterilization 15min；
F, solid fermentation: start the solid-state fermentation culture medium 8～10min of mechanical agitation oar whipping step e, its
Purpose is to be discharged by the chlorine dioxide of remaining in culture medium, in order to avoid damage solves starch in follow-up cultivation
Bacillus cell.Then, bacillus amyloliquefaciens B3 described in step b is inoculated into 5% inoculum concentration stirs
In solid-state fermentation culture medium after mixing, controlling fermentation temperature 34 DEG C, every 8h stirs once, every time stirring 8～
10min, ferment 96h, obtains solid fermentation wine with dregs；
G, it is dried: use 60 DEG C～the solid fermentation wine with dregs described in step f is dried water content 30% by 70 DEG C of hot blasts
Below；
H, pulverizing: use beater grinder the dried solid fermentation wine with dregs described in step g is ground into 60～
The powder of 70 mesh, i.e. obtains bacillus amyloliquefaciens B3 microbial inoculum, and its spore number reaches 22,000,000,000/g, bud
Spore rate reaches 99%.After microbial inoculum packaging, leave shady and cool being dried in and locate.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701619A (en) * | 2016-12-20 | 2017-05-24 | 湖南泰谷生物科技股份有限公司 | High-density fermentation method for bacillus amyloliquefaciens and preparation method of microbial agent of bacillus amyloliquefaciens |
| CN107836567A (en) * | 2017-11-22 | 2018-03-27 | 山东信得科技股份有限公司 | A kind of deoderizing feed addictive, preparation method, application method and evaluation method |
| CN110684691A (en) * | 2019-10-23 | 2020-01-14 | 陕西麦可罗生物科技有限公司 | Preparation process of microbial agent based on directional screening of microorganisms |
| CN112322562A (en) * | 2020-12-25 | 2021-02-05 | 广西壮族自治区畜牧研究所 | Solid culture medium of bacillus, preparation method thereof and bacillus culture method |
Citations (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002007520A2 (en) * | 2000-07-20 | 2002-01-31 | Ecolab Inc. | Antimicrobial composition useful for the treatment of bovine mastitis |
| JP2003039023A (en) * | 2001-07-31 | 2003-02-12 | Daiki:Kk | Method for recovering raw material from soiled sanitary goods |
| CN1597924A (en) * | 2004-07-22 | 2005-03-23 | 深圳职业技术学院 | Application of using chlorine dioxide as disinfecting agent for cultivating glossy ganoderma |
| CN101491290A (en) * | 2008-12-29 | 2009-07-29 | 贵州大学 | Method for producing livestock birds health cultivation green feed additive using distilled grain |
| CN101884301A (en) * | 2010-04-08 | 2010-11-17 | 浙江省农业科学院 | Method for preventing pollution in plant tissue culture |
| CN102031230A (en) * | 2010-08-17 | 2011-04-27 | 河北省农林科学院遗传生理研究所 | Bacillus amyloliquefacien and preparation method of polluted water reparation agent by using same |
| CN102191203A (en) * | 2011-04-13 | 2011-09-21 | 江南大学 | Bacillus amyloliquefaciens and method for producing chymosin by fermenting using same |
| CN102584451A (en) * | 2012-02-21 | 2012-07-18 | 山东省农业科学院农业资源与环境研究所 | Method for producing mushroom sticks by using bark waste as main material |
| CN102580129A (en) * | 2012-02-25 | 2012-07-18 | 何寒 | High-pressure-free and short-term normal pressure sterilizing method for cordyceps sinensis culture medium |
| CN102864100A (en) * | 2012-08-16 | 2013-01-09 | 北京雷力农用化学有限公司 | Bacillus amyloliquefaciens and application thereof |
| CN102960179A (en) * | 2011-08-29 | 2013-03-13 | 何寒 | Process for manufacturing liquid strain by using raw culture medium |
| CN103131648A (en) * | 2011-11-30 | 2013-06-05 | 河北农业大学 | Bacillus amyloliquefaciens and application of bacillus amyloliquefaciens in peanut rot disease preventive treatment |
| CN103255073A (en) * | 2012-02-15 | 2013-08-21 | 何寒 | Method for rapid propagation of high purity photosynthetic bacterium |
| CN104069521A (en) * | 2014-06-18 | 2014-10-01 | 新奥科技发展有限公司 | Method for governing pollution in micro-algae cultivation process |
| CN104152382A (en) * | 2014-08-08 | 2014-11-19 | 中国农业大学 | Bacillus amyloliquefaciens and application thereof |
| CN104232528A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus amyloliquefaciens preparation |
| CN104673728A (en) * | 2015-03-20 | 2015-06-03 | 济南航晨生物科技有限公司 | In-situ solid fermentation method for agricultural bacillus microbial inoculum |
| CN104694430A (en) * | 2015-03-09 | 2015-06-10 | 珠海真绿色技术有限公司 | Bacillus amyloliquefaciens subsp. plantarum ZFH-3 and application thereof |
| CN104782961A (en) * | 2015-04-13 | 2015-07-22 | 凤阳县兴科农业生态发展有限公司 | Apple pomace enzymatic bioprotein perilla leaf summer-heat-relieving pig feed and preparation method thereof |
| CN104782882A (en) * | 2015-04-13 | 2015-07-22 | 凤阳县兴科农业生态发展有限公司 | Apple pomace enzymatic bioprotein pollen pini sleep-promoting pig feed and preparation method thereof |
| CN104830711A (en) * | 2015-03-09 | 2015-08-12 | 浙江农林大学 | Solid fermentation process of bacillus amyloliquefaciens feed additive |
| CN104962492A (en) * | 2015-06-18 | 2015-10-07 | 中国农业大学 | Bacillus amyloliquefaciens, method for preparing solid inoculant thereof and application of solid inoculant |
| CN105385643A (en) * | 2015-12-27 | 2016-03-09 | 中国热带农业科学院南亚热带作物研究所 | Compound microbial agent for preventing and treating banana wilt disease and prevention and treatment method |
-
2016
- 2016-04-27 CN CN201610268604.3A patent/CN105886430A/en active Pending
Patent Citations (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002007520A2 (en) * | 2000-07-20 | 2002-01-31 | Ecolab Inc. | Antimicrobial composition useful for the treatment of bovine mastitis |
| JP2003039023A (en) * | 2001-07-31 | 2003-02-12 | Daiki:Kk | Method for recovering raw material from soiled sanitary goods |
| CN1597924A (en) * | 2004-07-22 | 2005-03-23 | 深圳职业技术学院 | Application of using chlorine dioxide as disinfecting agent for cultivating glossy ganoderma |
| CN101491290A (en) * | 2008-12-29 | 2009-07-29 | 贵州大学 | Method for producing livestock birds health cultivation green feed additive using distilled grain |
| CN101884301A (en) * | 2010-04-08 | 2010-11-17 | 浙江省农业科学院 | Method for preventing pollution in plant tissue culture |
| CN102031230A (en) * | 2010-08-17 | 2011-04-27 | 河北省农林科学院遗传生理研究所 | Bacillus amyloliquefacien and preparation method of polluted water reparation agent by using same |
| CN102191203A (en) * | 2011-04-13 | 2011-09-21 | 江南大学 | Bacillus amyloliquefaciens and method for producing chymosin by fermenting using same |
| CN102960179A (en) * | 2011-08-29 | 2013-03-13 | 何寒 | Process for manufacturing liquid strain by using raw culture medium |
| CN103131648A (en) * | 2011-11-30 | 2013-06-05 | 河北农业大学 | Bacillus amyloliquefaciens and application of bacillus amyloliquefaciens in peanut rot disease preventive treatment |
| CN103255073A (en) * | 2012-02-15 | 2013-08-21 | 何寒 | Method for rapid propagation of high purity photosynthetic bacterium |
| CN102584451A (en) * | 2012-02-21 | 2012-07-18 | 山东省农业科学院农业资源与环境研究所 | Method for producing mushroom sticks by using bark waste as main material |
| CN102580129A (en) * | 2012-02-25 | 2012-07-18 | 何寒 | High-pressure-free and short-term normal pressure sterilizing method for cordyceps sinensis culture medium |
| CN102864100A (en) * | 2012-08-16 | 2013-01-09 | 北京雷力农用化学有限公司 | Bacillus amyloliquefaciens and application thereof |
| CN104069521A (en) * | 2014-06-18 | 2014-10-01 | 新奥科技发展有限公司 | Method for governing pollution in micro-algae cultivation process |
| CN104152382A (en) * | 2014-08-08 | 2014-11-19 | 中国农业大学 | Bacillus amyloliquefaciens and application thereof |
| CN104232528A (en) * | 2014-08-29 | 2014-12-24 | 湖北省生物农药工程研究中心 | Process for preparing viable bacillus amyloliquefaciens preparation |
| CN104694430A (en) * | 2015-03-09 | 2015-06-10 | 珠海真绿色技术有限公司 | Bacillus amyloliquefaciens subsp. plantarum ZFH-3 and application thereof |
| CN104830711A (en) * | 2015-03-09 | 2015-08-12 | 浙江农林大学 | Solid fermentation process of bacillus amyloliquefaciens feed additive |
| CN104673728A (en) * | 2015-03-20 | 2015-06-03 | 济南航晨生物科技有限公司 | In-situ solid fermentation method for agricultural bacillus microbial inoculum |
| CN104782961A (en) * | 2015-04-13 | 2015-07-22 | 凤阳县兴科农业生态发展有限公司 | Apple pomace enzymatic bioprotein perilla leaf summer-heat-relieving pig feed and preparation method thereof |
| CN104782882A (en) * | 2015-04-13 | 2015-07-22 | 凤阳县兴科农业生态发展有限公司 | Apple pomace enzymatic bioprotein pollen pini sleep-promoting pig feed and preparation method thereof |
| CN104962492A (en) * | 2015-06-18 | 2015-10-07 | 中国农业大学 | Bacillus amyloliquefaciens, method for preparing solid inoculant thereof and application of solid inoculant |
| CN105385643A (en) * | 2015-12-27 | 2016-03-09 | 中国热带农业科学院南亚热带作物研究所 | Compound microbial agent for preventing and treating banana wilt disease and prevention and treatment method |
Non-Patent Citations (4)
| Title |
|---|
| BEUCHAT, LR: "《JOURNAL OF FOOD PROTECTION》", 《LETHALITY OF CHLORINE, CHLORINE DIOXIDE, AND A COMMERCIAL FRUIT AND VEGETABLE SANITIZER TO VEGETATIVE CELLS AND SPORES OF BACILLUS CEREUS AND SPORES OF BACILLUS THURINGIENSIS》 * |
| CARDOSO, JC等: "《Micropropagation of gerbera using chlorine dioxide (ClO2) to sterilize the culture medium》", 《IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-PLANT》 * |
| PEREZ, J等: "《Activity of selected oxidizing microbicides against the spores of Clostridium difficile: Relevance to environmental control》", 《AMERICAN JOURNAL OF INFECTION CONTROL》 * |
| RAMFREZ-OROZCO, M: "《Debaryomyces hansenii growth in nonsterile seawater ClO2-peptone-containing medium》", 《CANADIAN JOURNAL OF MICROBIOLOGY》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701619A (en) * | 2016-12-20 | 2017-05-24 | 湖南泰谷生物科技股份有限公司 | High-density fermentation method for bacillus amyloliquefaciens and preparation method of microbial agent of bacillus amyloliquefaciens |
| CN106701619B (en) * | 2016-12-20 | 2019-12-10 | 泰谷生态科技集团股份有限公司 | high-density fermentation method of bacillus amyloliquefaciens and preparation method of microbial inoculum thereof |
| CN107836567A (en) * | 2017-11-22 | 2018-03-27 | 山东信得科技股份有限公司 | A kind of deoderizing feed addictive, preparation method, application method and evaluation method |
| CN110684691A (en) * | 2019-10-23 | 2020-01-14 | 陕西麦可罗生物科技有限公司 | Preparation process of microbial agent based on directional screening of microorganisms |
| CN112322562A (en) * | 2020-12-25 | 2021-02-05 | 广西壮族自治区畜牧研究所 | Solid culture medium of bacillus, preparation method thereof and bacillus culture method |
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