CN106148247A - The fermentation method for producing of Ke Liben series bacillus and culture medium - Google Patents
The fermentation method for producing of Ke Liben series bacillus and culture medium Download PDFInfo
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Abstract
The invention provides the fermentation method for producing of a kind of Ke Liben series bacillus, Ke Liben series bacillus actication of culture, at first order seed liquid culture medium and secondary liquid culture medium culturing with expanding propagation, obtain strong thalline and spore, secondary seed solution is inoculated on three grades of fluid mediums of compound carbon source and the formation of nitrogen source, makes the fast-growth of Ke Liben series bacillus and produce spore.Three grades of fluid mediums include following masses percentage composition component: glucose 2.00～3.00%, Semen Maydis powder 0.50～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03～0.10%, calcium chloride 0.07～0.20%, surplus is water.In the fermentation liquid obtained, viable count is 10 50 hundred million CFU/ml, and after spray drying, obtaining the yield of mycopowder is 10% 15%.Technical scheme is used finally to obtain the Ke Liben series bacillus powder that spore content is higher.
Description
Technical field
The present invention relates to technical field of microbial fermentation, be specifically related to Ke Liben series bacillus fermentation method for producing and
The culture medium of Ke Liben series bacillus.
Background technology
Ke Liben series bacillus (Hypocrea virens) is a kind of heavy responsibilities of government suppressing multiple Soil-born plant pathogenic fungi
Raw bacterium, colony growth is fast, diameter 7～9.5cm after cultivating 4 days, aerial hyphae frizzle shape, white or Lycoperdon polymorphum Vitt.Conidium is rich
Richness, is bordering on open and flat shape and is covered in flat board.Ke Liben series bacillus, as the main mechanism of biocontrol of plant disease, has been got over
Get over and paid attention to by various countries phytopathologist.Hyperparasite is as the important factor of Biological control, for preventing and treating phytopathy
Evil has important function.
In prior art, the report about Ke Liben series bacillus fermentation technology is less, only Patent No. CN
The patent of invention of 103756931 A describes the discovery about Ke Liben series bacillus and the related content of application, Ke Liben
Series bacillus cultural method and culture medium are only the activation culture to Ke Liben series bacillus and activation medium two aspect
On the books, the culture medium not used with regard to Ke Liben series bacillus fermentation process and each stage of fermenting is on the books.
Ke Liben series bacillus activation culture method cannot meet the application of this area prevention plant disease, and traditional
Fungi fermentation method is capable of industrialized production, but it is long to there is fermentation time, and cost is high, the problems such as yield is low, it is impossible to meet
The existing application preventing plant disease.
Summary of the invention
In view of this, it is an object of the invention to the fermentation method for producing obtaining optimizing, it is possible to obtain a kind of more efficient
Control device, the Ke Liben series bacillus powder that final acquisition spore content is higher.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the fermentation method for producing of a kind of Ke Liben series bacillus, comprise the following steps:
1) by Ke Liben series bacillus actication of culture, it is inoculated in level liquid culture medium and carries out one-level cultivation, obtain
Primary seed solution；
2) by described step 1) primary seed solution that obtains is inoculated into secondary liquid culture medium and carries out second order fermentation cultivation,
To secondary seed solution；
3) by described step 2) secondary seed solution that obtains is inoculated into three grades of fluid mediums and carries out three grade fermemtation cultivation, dense
Division, from rear, obtains Ke Liben series bacillus mycopowder；
Described three grades of fluid mediums include following masses percentage composition component: glucose 2.00～3.00%, Semen Maydis powder
0.50～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03～0.10%, calcium chloride 0.07～0.20%, remaining
Amount is water.
Preferably, described step 1) in time of cultivating of one-level be 28～32h, the temperature that one-level is cultivated is 28～32 DEG C.
Preferably, described step 1) in one-level cultivate and must carry out illumination cultivation, the light intensity 3000 of described illumination cultivation～
4000xl。
Preferably, described step 1) in one-level incubation ventilate, the program of described ventilation is particularly as follows: enter training
Supporting in 24h, ventilation is 10～20m3/ h, after cultivating 24h, ventilation 15～24m3/h；
Preferably, the inoculum concentration of described cultivation is 1.5～2.5%.
Preferably, the original ph that described one-level is cultivated is 7.0～8.0.
Preferably, described step 1) in one-level cultivate and carry out under conditions of stirring, the frequency of described stirring is 1～2
Secondary/h, the time of stirring is 10～20min every time, and the rotating speed of described stirring is 150～300r/min.
Preferably, described step 2) in time of cultivating of second order fermentation be 45～52h, second order fermentation cultivation temperature be 30～
35℃。
Described second order fermentation is cultivated and is carried out under light illumination, and the light intensity of described illumination is 4000～5000xl.
Preferably, described second order fermentation incubation is ventilated, and the program of described ventilation is particularly as follows: enter second order fermentation
After cultivation within 24h, ventilation controls 100～180m3/ h, after cultivating 24h, ventilation is 200～260m3/h。
Preferably, the inoculum concentration that described second order fermentation is cultivated is 7～15%.
Preferably, the initial pH that described second order fermentation is cultivated is 7.0～8.0.
Preferably, described step 2) in second order fermentation cultivate and carry out under conditions of stirring, the frequency of described stirring is 1
～2 times/h, the time of stirring is 10～20min (inventor needs to confirm) every time, and the rotating speed of described stirring is 150～300r/
min。
Preferably, described step 3) time of cultivating of three grade fermemtation is 62～67h, the temperature 28～32 that three grade fermemtation is cultivated
℃。
Described three grade fermemtation cultivate carry out under light illumination, described illumination light intensity be 5000～7000xl.
Preferably, described three grade fermemtation incubation is ventilated, and the program of described ventilation is particularly as follows: enter three grade fermemtation
After cultivation within 36h, ventilation controls 1800～2300m3/ h, after cultivating 48h, ventilation is 800～1200m3/h。
Preferably, the inoculum concentration that described three grade fermemtation is cultivated is 7～15%.
Preferably, the original ph that described three grade fermemtation is cultivated is 7.0～8.0.
Preferably, described step 3) in three grade fermemtation cultivate and carry out under conditions of stirring, the frequency of described stirring is 1
～2 times/h, the time of stirring is 10～20min every time, and the rotating speed of described stirring is 150～300r/min.
Preferably, it is characterised in that described level liquid culture medium includes following masses percentage composition component: sucrose 2～
5%, sodium nitrate 0.1～0.2%, dipotassium hydrogen phosphate 0.08～0.2%, magnesium sulfate 0.03～0.1%, potassium chloride 0.02～
0.1%, iron sulfate 0.001～0.003%, surplus is water；It is furthermore preferred that described level liquid culture medium includes that following percentage contains
Amount component: sucrose 3%, sodium nitrate 0.15%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, iron sulfate
0.001%, surplus is water.
Preferably, described secondary liquid culture medium includes following masses percentage composition component: sucrose 1.00～2.00%, Portugal
Grape sugar 1.00～2.00%, analysis for soybean powder 0.70～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03～
0.10%, calcium chloride 0.07～0.20%, surplus is water；It is furthermore preferred that described secondary liquid culture medium includes that following percentage contains
Amount component: sucrose 1.5%, glucose 1.5%, analysis for soybean powder 1%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.05%, calcium chloride
0.1%, surplus is water.
Present invention also offers the fermenting and producing culture medium of a kind of Ke Liben series bacillus, including following masses percentage
Content component: glucose 2.00～3.00%, Semen Maydis powder 0.50～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate
0.03～0.10%, calcium chloride 0.07～0.20%, surplus is water.
Preferably, including following masses percentage composition component: glucose 2.5%, Semen Maydis powder 1%, ammonium sulfate 0.05%, phosphorus
Acid hydrogen dipotassium 0.05%, calcium chloride 0.1%, surplus is water.
The invention provides the fermentation method for producing of a kind of Ke Liben series bacillus, by Ke Liben series bacillus strain
Activation, carry out cultivating with expanding propagation in first order seed liquid culture medium and secondary liquid culture medium, obtain strong thalline and
A certain amount of spore, is inoculated into secondary seed solution on three grades of fluid mediums that compound carbon source and nitrogen source are formed, in making gram
The fast-growth of this series bacillus and product spore.In the fermentation liquid obtained, viable count is 10-50 hundred million CFU/ml, is spray-dried
After, the yield obtaining mycopowder is 10%-15%.As can be seen here, technical scheme is used finally to obtain spore content relatively
High Ke Liben series bacillus powder, solves the technical problem of the present invention.
Accompanying drawing explanation
Fig. 1 is three grade fermemtation bacterium solution process chart in the embodiment of the present invention.
Culture presevation
Ke Liben series bacillus (Paenibacillus kribbensis), contains in Chinese microorganism strain preservation pipe
Reason committee's common micro-organisms center, address is China. Beijing. and Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese science
Institute of Micro-biology of institute, preservation mechanism is called for short: CGMCC, and preservation date is on August 13rd, 2013, deposit number CGMCC No.7996,
Strain number: TRCC82001.
Detailed description of the invention
The invention provides the fermentation method for producing of a kind of Ke Liben series bacillus, comprise the following steps:
1) by Ke Liben series bacillus actication of culture, it is inoculated in level liquid culture medium and carries out one-level cultivation, obtain
Primary seed solution；
2) by described step 1) primary seed solution that obtains is inoculated into secondary liquid culture medium and carries out second order fermentation cultivation,
To secondary seed solution；
3) by described step 2) secondary seed solution that obtains is inoculated into three grades of fluid mediums and carries out three grade fermemtation cultivation, dense
Division, from rear, obtains Ke Liben series bacillus mycopowder；
Described three grades of fluid mediums include following masses percentage composition component: glucose 2.00～3.00%, Semen Maydis powder
0.50～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03～0.10%, calcium chloride 0.07～0.20%, remaining
Amount is water.
The invention provides the fermentation method for producing of a kind of Ke Liben series bacillus, by Ke Liben series bacillus strain
Activation, carry out cultivating with expanding propagation in first order seed liquid culture medium and secondary liquid culture medium, obtain strong thalline and
A certain amount of spore, is inoculated on the three-stage culture medium formed by the carbon source being combined and nitrogen source three grades carried out by secondary seed solution
Fermentation culture, makes the fast-growth of Ke Liben series bacillus and produces spore.
The present invention, by Ke Liben series bacillus actication of culture, obtains the Ke Liben series bacillus strain of activation.This
The bright method to described activation is not particularly limited, and uses the technical scheme of activation well known to those skilled in the art.?
In embodiments of the invention, described Ke Liben series bacillus strain provides for Jiangxi Tianren Ecology Co., Ltd..
In the present invention, the culture medium of described activation is preferably the solid Czapek's medium II of improvement.
In the present invention, the formula of described solid Czapek's medium II sees the special of Application No. CN201310643992.5
Profit.
Obtaining the Ke Liben series bacillus of activation, the Ke Liben series bacillus of described activation is inoculated into one by the present invention
Level seed fluid medium carries out one-level cultivation, obtains primary seed solution.
In the present invention, the time that described one-level is cultivated is preferably 28～32h, more preferably 30 DEG C.Described one-level is cultivated
Temperature be preferably 28～32 DEG C, more preferably 30 DEG C.Described one-level is cultivated and is preferably carried out under illumination condition, described light
According to light intensity be preferably 3000～4000lx, more preferably 3500lx.
In the present invention, described one-level cultivate preferably stirring under conditions of carry out, the frequency of described stirring be preferably 1～
2 times/h, the time of stirring is preferably 10～20min every time.The rotating speed of described stirring is 150～300r/min.
In the present invention, described one-level incubation is preferably ventilated, and the program of described ventilation is particularly preferred as: enter
Cultivating in 24h, ventilation is 10～20m3/ h, more preferably 15m3/h；Enter and cultivate after 24h, ventilation be preferably 15～
24m3/ h, more preferably 20m3/h。
In the present invention, the gaseous species of described ventilation is preferably filtrated air.
In the present invention, the inoculum concentration that described one-level is cultivated is preferably 1.5～2.5%, more preferably 2%.
In the present invention, the original ph that described one-level is cultivated is 7.0～8.0, more preferably 7.5.
In the present invention, described first order seed liquid culture medium preferably includes the component of following masses percentage composition: sucrose 2
～5%, sodium nitrate 0.1～0.2%, dipotassium hydrogen phosphate 0.08～0.2%, magnesium sulfate 0.03～0.1%, potassium chloride 0.02～
0.1%, iron sulfate 0.001～0.003%, surplus is water；More preferably sucrose 3%, sodium nitrate 0.15%, dipotassium hydrogen phosphate
0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, iron sulfate 0.001%, surplus is water.The present invention source to said components
It is not particularly limited, uses the medicine for preparing culture medium well-known to those skilled in the art.
After obtaining primary seed solution, described primary seed solution is inoculated in secondary liquid culture medium and carries out two grades by the present invention
Fermentation culture, obtains secondary seed solution.
The method of described inoculation is not particularly limited by the present invention, the inoculation known to described in employing those skilled in the art
Technical scheme.
In the present invention, the inoculum concentration that described second order fermentation is cultivated is preferably 7～15%, more preferably 10%.
In the present invention, the time that described second order fermentation is cultivated is preferably 45～52h, more preferably 48h.Described two grades
The temperature of fermentation culture is preferably 30～35 DEG C, more preferably 32 DEG C.Described second order fermentation is cultivated preferably to enter under illumination condition
OK, the light intensity of described illumination is preferably 4000～5000lx, more preferably 4500lx.
In the present invention, described second order fermentation incubation is preferably carried out under aeration condition, the program tool of described ventilation
Body is preferably: entering after second order fermentation is cultivated within 24h, ventilation controls 100～180m3/ h, more preferably 150m3/h；Training
After supporting 24h, ventilation is 200～260m3/H, more preferably 250m3/h。
In the present invention, the gaseous species of described ventilation is preferably filtrated air.
In the present invention, the initial pH that described second order fermentation is cultivated is preferably 7.0～8.0, more preferably 7.5.
In the present invention, described second order fermentation is cultivated and is preferably carried out under conditions of stirring, and the frequency of described stirring is preferred
Being 1～2 time/h, the time of stirring is 10～20min every time.The rotating speed of described stirring is 150～300r/min.
In the present invention, described secondary seed liquid culture medium preferably includes following masses percentage composition component: sucrose
1.00～2.00%, glucose 1.00～2.00%, analysis for soybean powder 0.70～1.50%, ammonium sulfate 0.03～0.10%, phosphoric acid hydrogen
Dipotassium 0.03～0.10%, calcium chloride 0.07～0.20%, surplus is water, more preferably sucrose 1.5%, glucose 1.5%, yellow
Semen Glycines powder 1%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.05%, calcium chloride 0.1%, surplus is water.
After obtaining secondary seed solution, described secondary seed solution is inoculated into three grades of fluid mediums and carries out three grades by the present invention
Ferment is cultivated, and obtains three grade fermemtation bacterium solution.
In the present invention, the time that described three grade fermemtation is cultivated is preferably 62～67h, more preferably 64h.Send out for described three grades
The temperature that ferment is cultivated is preferably 28～32 DEG C, more preferably 30 DEG C.Described three grade fermemtation is cultivated preferably under the conditions of illumination cultivation
Carrying out, the light intensity of described illumination is preferably 5000～7000lx, more preferably 6000lx.
In the present invention, described three grade fermemtation incubation is ventilated, and the program of described ventilation is specific as follows: enter three
After level fermentation culture within 36h, ventilation controls 1800～2300m3/ h, more preferably 2000m3/h；After cultivating 48h, ventilation
Amount is preferably 800～1200m3/ h, more preferably 1000m3/h。
In the present invention, described three grade fermemtation is cultivated between 36-48, and ventilation gradually decreases, and in reverse V-shaped, enters when 42h
Row anoxia operates, and is gradually increased ventilation afterwards.The effect of described anoxia operation is to promote mycelia under conditions of suitable anoxia
Body is changed into brood-gemma greatly.
In the present invention, the gaseous species of described ventilation is preferably filtrated air.
In the present invention, the inoculum concentration that described three grade fermemtation is cultivated is preferably 7～15%, more preferably 10%.
In the present invention, the original ph that described three grade fermemtation is cultivated is 7.0～8.0, more preferably 7.5.
In the present invention, described three grade fermemtation is cultivated and is preferably carried out under conditions of stirring, and the frequency of described stirring is preferred
Being 1～2 time/h, the time of stirring is preferably 10～20min every time.The rotating speed of described stirring is 150～300r/min.
After three grade fermemtation is cultivated, the three grade fermemtation culture fluid obtained preferably is separated by the present invention, obtains supernatant and concentration
Liquid.
In the present invention, described centrifugal rotating speed is that 10000-15000r/min is more preferably 12000～13000r/min.
In the present invention, described centrifugal equipment is not particularly limited, and uses well-known to those skilled in the art centrifugal
Machine.In embodiments of the present invention, described centrifugal equipment is butterfly centrifugal machine.
In the present invention, the preferred spray-dried tower of described concentrated solution is dried, and finally gives Ke Liben series bacillus
Mycopowder.
In the present invention, described dry temperature is preferably 35 DEG C～40 DEG C, more preferably 37 DEG C.
In the present invention, obtain without fermented liquid product after described supernatant is packaged.
Present invention also offers the fermenting and producing culture medium of a kind of Ke Liben series bacillus, including following masses percentage
Content component: glucose 2.00～3.00%, Semen Maydis powder 0.50～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate
0.03～0.10%, calcium chloride 0.07～0.20%, surplus is water；More preferably include following masses percentage composition component: Portugal
Grape sugar 2.5%, Semen Maydis powder 1%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.05%, calcium chloride 0.1%, surplus is water.
The fermenting and producing culture medium of a kind of Ke Liben series bacillus that the present invention provides, described culture medium uses compound
Nitrogen source and carbon source and nutrient form, and by optimizing each component proportion, make thalline produce spore and the metabolism product of rich content
Thing.
Fermentation method for producing and the culture medium of the Ke Liben series bacillus provided the present invention below in conjunction with embodiment are entered
Row detailed description, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
The Ke Liben series bacillus strain that Jiangxi Tianren Ecology Co., Ltd. provides is in the solid Cha Shi training of improvement
Support the inoculum concentration inoculating 1% on the solid slope of base II, cultivate 3 days under the conditions of 30 DEG C, strain is activated.By activation
Strain is inoculated in level liquid culture medium at 30 DEG C of CMC model 30h according to the inoculum concentration of 2%, and incubation adds illumination,
Light intensity is 3000lx, carries out one-level cultivation, and incubation is passed through filtrated air, enters and cultivates in 24h, and ventilation is 18m3/ h,
After cultivating 24h, ventilation 20m3/ h, incubation need to be carried out under conditions of stirring simultaneously, and interior stirring per hour once, is stirred every time
Mixing lasting 20min, the rotating speed of stirring is 150～300r/min, and level liquid culture medium prescription is sucrose 3%, sodium nitrate
0.15%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, iron sulfate 0.001%, surplus is water, cultivates
Original ph be 7.5.After terminating one-level cultivation, it is inoculated into secondary liquid by obtaining primary seed solution according to the inoculum concentration of 10%
In culture medium, secondary liquid culture medium includes sucrose 1.5%, glucose 1.5%, analysis for soybean powder 1%, ammonium sulfate 0.05%, phosphoric acid
Hydrogen dipotassium 0.05%, calcium chloride 0.1%, surplus is water, and the original ph of cultivation is 7.5, cultivates 48h, training under the conditions of 32 DEG C
Foster process needs the illumination of 5000lx, enter two grades cultivate after within 24h, be passed through filtrated air 150m3/ h, after cultivating 24h, ventilation
250m it is transferred in amount3/ h, stirs per hour in two grades of incubation 2 times, stirs 10min every time.After terminating two grades of cultivations, will obtain
Obtaining secondary seed solution to be inoculated on three grades of fluid mediums according to the inoculum concentration of 10%, secondary liquid culture medium includes glucose
2.5%, Semen Maydis powder 1%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.05%, calcium chloride 0.1%, surplus is water, cultivation initial
PH value is 7.5；Under the conditions of 30 DEG C, 64h is cultivated in three grade fermemtation, and in incubation, the intensity control of illumination is 5000lx, enters three
After level fermentation culture within 36h, ventilation controls at 2000m3/ h, after cultivating 48h, ventilation is 1000m3/h；Stir per hour
2 times, stirring continues 15min (inventor needs to confirm) every time.After obtaining three grade fermemtation bacterium solution, butterfly centrifugal machine is used to exist
It is centrifuged under the conditions of 10000r/min, obtains supernatant and concentrated solution, obtain without fermented liquid product after supernatant is packaged, by dense
37 DEG C of the spray-dried tower of contracting liquid is dried, and obtains the mycopowder of Ke Liben series bacillus.
Embodiment 2
The Ke Liben series bacillus strain that Jiangxi Tianren Ecology Co., Ltd. provides is in the solid Cha Shi training of improvement
Support the inoculum concentration inoculating 1% on the solid slope of base II, cultivate 3 days under the conditions of 30 DEG C, strain is activated.By activation
Strain is inoculated in level liquid culture medium at 32 DEG C of CMC model 28h according to the inoculum concentration of 1.5%, and incubation is under light illumination
Carrying out, light intensity is 4000lx, carries out one-level cultivation, and incubation is passed through filtrated air, enters and cultivates in 24h, and ventilation is
10m3/ h, after cultivating 24h, ventilation 15m3/ h, incubation need to be carried out under conditions of stirring simultaneously, per hour in stirring once,
Every time stirring continues 20min, and the rotating speed of described stirring is 150～300r/min, level liquid culture medium prescription be sucrose 5%,
Sodium nitrate 0.1%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.03%, potassium chloride 0.1%, iron sulfate 0.001%, surplus is water,
The original ph cultivated is 7.0.After terminating one-level cultivation, it is inoculated into two grades by obtaining primary seed solution according to the inoculum concentration of 7%
On fluid medium, secondary liquid culture medium includes sucrose 1.00%, glucose 2.00%, analysis for soybean powder 0.70%, ammonium sulfate
0.10%, dipotassium hydrogen phosphate 0.03%, calcium chloride 0.20%, surplus is water, and the original ph of cultivation is 8.0,30 DEG C of conditions
Lower cultivation 45h, incubation needs the illumination of 45000lx, enter two grades cultivate after within 24h, be passed through filtrated air 180m3/ h,
After cultivating 24h, ventilation is transferred to 200m3/ h, stirs per hour in two grades of incubation 2 times, stirs 10min every time.Terminate
After two grades are cultivated, it is inoculated into obtaining secondary seed solution on three grades of fluid mediums according to the inoculum concentration of 10%, three grades of liquid trainings
Foster base includes glucose 2.00%, Semen Maydis powder 1.50%, ammonium sulfate 0.03%, dipotassium hydrogen phosphate 0.10%, calcium chloride 0.07%,
Surplus is water, and the original ph of cultivation is 7.5；Under the conditions of 32 DEG C, 62h, the light intensity of illumination in incubation are cultivated in three grade fermemtation
Controlling is 7000lx, enters after three grade fermemtation is cultivated within 36h, and ventilation controls at 2300m3/ h, after cultivating 48h, ventilation
For 1200m3/h；Stirring 2 times per hour, stirring continues 15min (inventor needs to confirm) every time.After obtaining three grade fermemtation bacterium solution,
Use butterfly centrifugal machine centrifugal under the conditions of 15000r/min, obtain supernatant and concentrated solution, after supernatant is packaged, obtain nothing
Fermented liquid product, is dried 40 DEG C of spray-dried for concentrated solution tower, obtains the mycopowder of Ke Liben series bacillus.
Embodiment 3
The Ke Liben series bacillus strain that Jiangxi Tianren Ecology Co., Ltd. provides is in the solid Cha Shi training of improvement
Support the inoculum concentration inoculating 1% on the solid slope of base II, cultivate 3 days under the conditions of 30 DEG C, strain is activated.By activation
Strain is inoculated in level liquid culture medium at 28 DEG C of CMC model 32h according to the inoculum concentration of 2.5%, and incubation is under light illumination
Carrying out, light intensity is 3500lx, carries out one-level cultivation, and incubation is passed through filtrated air, enters and cultivates in 24h, and ventilation is
20m3/ h, after cultivating 24h, ventilation 22m3/ h, incubation need to be carried out under conditions of stirring simultaneously, per hour in stirring once,
Every time stirring continues 20min, and the rotating speed of described stirring is 150～300r/min, level liquid culture medium prescription be sucrose 2%,
Sodium nitrate 0.2%, dipotassium hydrogen phosphate 0.08%, magnesium sulfate 0.1%, potassium chloride 0.02%, iron sulfate 0.003%, surplus is water,
The original ph cultivated is 8.0.After terminating one-level cultivation, it is inoculated into two grades by obtaining primary seed solution according to the inoculum concentration of 15%
On fluid medium, secondary liquid culture medium includes sucrose 2.00%, glucose 1.00%, analysis for soybean powder 1.50%, ammonium sulfate
0.03%, dipotassium hydrogen phosphate 0.10%, calcium chloride 0.07%, surplus is water, and the original ph of cultivation is 7.5,35 DEG C of conditions
Lower cultivation 45h, incubation needs the illumination of 4000lx, enter two grades cultivate after within 24h, be passed through filtrated air 180m3/ h, training
After supporting 24h, ventilation is transferred to 200m3/ h, stirs per hour in two grades of incubation 2 times, stirs 10min every time.Terminate two
After level is cultivated, it is inoculated into obtaining secondary seed solution on three grades of fluid mediums according to the inoculum concentration of 10%, three grades of liquid cultures
Base includes glucose 2.00～3.00%, Semen Maydis powder 0.50～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03
～0.10%, calcium chloride 0.07～0.20%, surplus is water, and the original ph of cultivation is 7.5；Three grade fermemtation under the conditions of 28 DEG C
Cultivating 67h, in incubation, the intensity control of illumination is 6000lx, enters after three grade fermemtation is cultivated within 36h, and ventilation controls
At 1800m3/ h, after cultivating 48h, ventilation is 800m3/h；Stirring 2 times per hour, stirring continues 20min every time.Obtain three grades
After zymocyte liquid, using butterfly centrifugal machine centrifugal under the conditions of 12000r/min, obtain supernatant and concentrated solution, supernatant is through bag
Obtain after dress, without fermented liquid product, being dried by 35 DEG C of spray-dried for concentrated solution tower, obtaining the bacterium of Ke Liben series bacillus
Powder.
In the fermentation liquid obtained in embodiment 1-3, viable count is 10-50 hundred million CFU/ml, the mycopowder being dried to obtain and fermentation liquid
Ratio be mycopowder yield, the mycopowder yield being computed obtaining in embodiment 1-3 is 10%-15%.
As seen from the above embodiment, use suitable carbon-nitrogen ratio, with high nitrogen content in prepared by primary seed solution, promote
The thalline that strain is concentrated grows more strong thalline on producing, and in secondary seed solution produces, carbon-nitrogen ratio thalline produces suitable
Preferably, having strong mycelia can produce more spore, spore germination is in bacterium solution simultaneously, can cultivate rank in three grade fermemtation
The thalline of greater amount in Duan；Optimum abiotic factor of influence is provided, optimizes fermentation culture parameter aborning, more efficient
Control device, final concentration dries out mycopowder.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. the fermentation method for producing of Yi Zhong Ke Liben series bacillus, it is characterised in that comprise the following steps:
1) by Ke Liben series bacillus actication of culture, it is inoculated in level liquid culture medium and carries out one-level cultivation, obtain one-level
Seed liquor；
2) by described step 1) primary seed solution that obtains is inoculated into secondary liquid culture medium and carries out second order fermentation cultivation, obtains two
Level seed liquor；
3) by described step 2) secondary seed solution that obtains is inoculated into three grades of fluid mediums and carries out three grade fermemtation cultivation, obtains gram
In this series bacillus mycopowder；
Described three grades of fluid mediums include following masses percentage composition component: glucose 2.00～3.00%, Semen Maydis powder 0.50
～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03～0.10%, calcium chloride 0.07～0.20%, surplus is
Water.
Fermentation method for producing the most according to claim 1, it is characterised in that described step 1) in one-level cultivate time be
28～32h, the temperature that one-level is cultivated is 28～32 DEG C；
Described one-level is cultivated and is carried out under light illumination, and the light intensity of described illumination is 3000～4000lx；
Described step 1) in one-level incubation ventilate, the program of described ventilation is particularly as follows: enter and cultivate in 24h, logical
Tolerance is 10～20m3/ h, after cultivating 24h, ventilation is 15～24m3/h；
The inoculum concentration that described one-level is cultivated is 1.5～2.5%；
The original ph that described one-level is cultivated is 7.0～8.0；
Described step 1) in one-level cultivate stirring under conditions of carry out, the frequency of described stirring is 1～2 time/h, stirs every time
The time mixed is 10～20min, and the rotating speed of described stirring is 150～300r/min.
Method the most according to claim 1, it is characterised in that described step 2) in second order fermentation cultivate time be 45～
52h, two grades of temperature cultivated are 30～35 DEG C；
Described two grades of cultivations are carried out under light illumination, and the light intensity of described illumination is 4000～5000lx；
Described second order fermentation incubation is ventilated, the program of described ventilation particularly as follows: enter two grades cultivate after within 24h,
Ventilation controls 100～180m3/ h, after cultivating 24h, ventilation is 200～260m3/h；
The inoculum concentration that described second order fermentation is cultivated is 7～15%；
The initial pH that described second order fermentation is cultivated is 7.0～8.0；
Described step 2) in second order fermentation cultivate stirring under conditions of carry out, the frequency of described stirring is 1～2 time/h, often
The time of secondary stirring is 10～20min, and the rotating speed of described stirring is 150～300r/min.
Method the most according to claim 1, it is characterised in that described step 3) three grade fermemtation cultivate time be 62～
67h, the temperature 28 of cultivation～32 DEG C；
Described three grade fermemtation is cultivated and is carried out under light illumination, and the light intensity of described illumination is 5000～7000lx；
Described incubation is ventilated, and the program of described ventilation is particularly as follows: after entering three grade fermemtation cultivation within 36h, ventilate
Amount controls 1800～2300m3/ h, after cultivating 48h, ventilation is 800～1200m3/h；The inoculation that described three grade fermemtation is cultivated
Amount is 7～15%；
The original ph that described three grade fermemtation is cultivated is 7.0～8.0；
Described step 3) in three grade fermemtation cultivate stirring under conditions of carry out, the frequency of described stirring is 1～2 time/h, often
The time of secondary stirring is 10～20min, and the rotating speed of described stirring is 150～300r/min.
5. according to the fermentation method for producing described in Claims 1 to 4 any one, it is characterised in that described level liquid is cultivated
Base includes following masses percentage composition component: sucrose 2～5%, sodium nitrate 0.1～0.2%, dipotassium hydrogen phosphate 0.08～0.2%,
Magnesium sulfate 0.03～0.1%, potassium chloride 0.02～0.1%, iron sulfate 0.001～0.003%, surplus is water.
Fermentation method for producing the most according to claim 5, it is characterised in that described level liquid culture medium includes following hundred
Point content component: sucrose 3%, sodium nitrate 0.15%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, potassium chloride 0.05%, sulfur
Acid ferrum 0.001%, surplus is water.
7. according to the fermentation method for producing described in Claims 1 to 4 any one, it is characterised in that described secondary liquid is cultivated
Base includes following masses percentage composition component: sucrose 1.00～2.00%, glucose 1.00～2.00%, analysis for soybean powder 0.70～
1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03～0.10%, calcium chloride 0.07～0.20%, surplus is water.
Fermentation method for producing the most according to claim 1, it is characterised in that described secondary liquid culture medium includes following hundred
Point content component: sucrose 1.5%, glucose 1.5%, analysis for soybean powder 1%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.05%, chlorination
Calcium 0.1%, surplus is water.
9. the fermenting and producing culture medium of Yi Zhong Ke Liben series bacillus, it is characterised in that include following masses percentage composition
Component: glucose 2.00～3.00%, Semen Maydis powder 0.50～1.50%, ammonium sulfate 0.03～0.10%, dipotassium hydrogen phosphate 0.03
～0.10%, calcium chloride 0.07～0.20%, surplus is water.
Culture medium the most according to claim 9, it is characterised in that include following masses percentage composition component: glucose
2.5%, Semen Maydis powder 1%, ammonium sulfate 0.05%, dipotassium hydrogen phosphate 0.05%, calcium chloride 0.1%, surplus is water.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699530A (en) * | 2017-11-27 | 2018-02-16 | 周口师范学院 | A kind of fermentation of bacillus culture medium, fermentation process and application |
| CN109265461A (en) * | 2018-08-23 | 2019-01-25 | 华南农业大学 | A kind of Ke Liben series bacillus metabolin and its application in biological and ecological methods to prevent plant disease, pests, and erosion |
-
2016
- 2016-08-31 CN CN201610799389.XA patent/CN106148247A/en not_active Withdrawn
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699530A (en) * | 2017-11-27 | 2018-02-16 | 周口师范学院 | A kind of fermentation of bacillus culture medium, fermentation process and application |
| CN107699530B (en) * | 2017-11-27 | 2021-05-04 | 周口师范学院 | Bacillus fermentation medium, fermentation method and application |
| CN109265461A (en) * | 2018-08-23 | 2019-01-25 | 华南农业大学 | A kind of Ke Liben series bacillus metabolin and its application in biological and ecological methods to prevent plant disease, pests, and erosion |
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