CN104293721A - Method for high-density culture of bacillus subtilis - Google Patents
Method for high-density culture of bacillus subtilis Download PDFInfo
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- CN104293721A CN104293721A CN201410567431.6A CN201410567431A CN104293721A CN 104293721 A CN104293721 A CN 104293721A CN 201410567431 A CN201410567431 A CN 201410567431A CN 104293721 A CN104293721 A CN 104293721A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention relates to a method for high-density culture of bacillus subtilis. The method comprises the steps of (1) carrying out three-level seed activation by regarding bacillus subtilis as fermentation bacteria, wherein a activation culture medium comprises the components: 10g/L of peptone, 3g/L of beef powder, 5g/L of sodium chloride and 1g/L of glucose; (2) inoculating activated bacteria to a fermentation culture medium for culture, wherein the fermentation culture medium comprises the components: 10g/L of glucose, 15g/L of soybean cake powder, 0.3g/L of MnSO4, 5g/L of NaCl, 0.3g/L of K2HPO4, 5g/L of calcium carbonate; fermenting the components at a temperature of 30 DEG C under a stirring rotating speed of 200r/min for 48h to obtain high-density cultured bacillus subtilis. By improvement of culture medium prescriptions and fermentation conditions, viable count of bacillus subtilis is more than 8.9*10<9>cfu/mL, necessary conditions are provided for industrial fermentation of bacillus subtilis, and rate of production can be increased greatly.
Description
Technical field
The invention belongs to mushroom culture technique field, especially a kind of method of carrying out high-density culture on bacillus subtilis.
Background technology
Extracellular enzyme can be produced in the process that subtilis breeds in water body, it can by the remaining bait in water body and bed mud, the organic substance decomposing such as excrete wastes and plant and animal residues, first these organism are decomposed into small molecules, as higher fatty acid and polypeptide etc., then less molecular organic is decomposed into, as monose, amino acid, lower fatty acid and cyclic hydrocarbon etc., finally be decomposed into carbonic acid gas, nitrate, vitriol etc., eventually reduce the BOD in water, COD, make the nitrogen in water, ammonia, the concentration of sulfide and cultured water reduces, there is the effect of removing bed mud and alleviating body eutrophication, in addition the Multiple Classes of Antibiotics of secreted from bacillus and enzyme can suppress the growth of other pathogenic bacteria.
Subtilis is as additive used for aquiculture, and some researchs prove that it can promote breed variety accelerating growth, reduce feed coefficient, maintains intestinal microecology balance, strengthens animal body immunizing power, improve resistance against diseases and decreasing pollution etc.
The domestic research to subtilis at present mainly concentrates on the Enzymatic characteristic etc. of sieve bacterium, fungistatic effect and subtilis, and the fermentation technique for carrying out high-density culture on bacillus subtilis is then reported less.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, and propose a kind of method of carrying out high-density culture on bacillus subtilis.
The present invention solves its technical problem and takes following technical scheme to realize:
A method for carrying out high-density culture on bacillus subtilis, comprises step as follows:
(1) be that fermented bacterium carries out three grades of seed activations with subtilis,
Wherein, the component of activation medium comprises: peptone 10g/L, beef powder 3g/L, sodium-chlor 5g/L, glucose 1g/L;
Three grades of seed activation culture condition are: shaking table cultivation, pH value 7.5, temperature are 30 DEG C, time 24h;
(2) strain inoculation after activation is cultivated in fermention medium,
Wherein, the component of fermention medium comprises: glucose 10g/L, soybean cake powder 15g/L, MnSO
40.3g/L, NaCl5g/L, K
2hPO
40.3g/L, calcium carbonate 5g/L;
Fermentation condition is: after pH nature, culture temperature 30 DEG C, mixing speed 200r/min, incubation time 48h, obtain the subtilis of high-density culture.
And the inoculum size of three grades of seed activation cultivations is 8%v/v in described step (1).
And the inoculum size of fermentation culture is 8%v/v in described step (2).
And in described step (2) during fermentation culture, the oxygen-supply quantity of the 10L volume that often ferments is greater than 0.5m
3/ h.
Advantage of the present invention and positively effect are:
1, the present invention is by the improvement to culture medium prescription and fermentation condition, for improving biomass, the efficient condition that provides the foundation of raising product of subtilis.
2, after using the inventive method, the viable count of subtilis significantly improves, and reaches 8.9 × 10
9more than cfu/mL, than 1.8 × 10 before optimization
9cfu/mL viable count improves 4.9 times, improves output significantly.
Embodiment
Be further described the invention process below, following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.
A method for carrying out high-density culture on bacillus subtilis, comprises step as follows:
(1) be that fermented bacterium carries out three grades of seed activations with subtilis,
Wherein, the component of activation medium comprises: peptone 10g/L, beef powder 3g/L, sodium-chlor 5g/L, glucose 1g/L;
Three grades of seed activation culture condition are: shaking table cultivation, pH value 7.5, temperature are 30 DEG C, time 24h, inoculum size 8% (v/v).
(2) strain inoculation after activation is cultivated in fermention medium,
Wherein, the component of fermention medium comprises: glucose 10g/L, soybean cake powder 15g/L, MnSO
40.3g/L, NaCl5g/L, K
2hPO
40.3g/L, calcium carbonate 5g/L;
Fermentation condition is: pH nature, culture temperature 30 DEG C, mixing speed 200r/min, inoculum size are 8% (v/v), incubation time 48h, obtain the subtilis of high-density culture.
Example 1:
(1) seed culture:
The substratum that seed activation adopts is composed as follows: peptone 10g/L, beef powder 3g/L, sodium-chlor 5g/L, glucose 1g/L.
The condition of activation culture is: shaking table is cultivated; PH value 7.5; Temperature is 30 DEG C; Time 24h, inoculum size 8% (v/v).
(2) fermentation culture:
Be inoculated in fermention medium by the subtilis activated and cultivate, fermentation volume is 30L.
Described fermention medium is made up of following raw material: glucose 10g/L, soybean cake powder 15g/L, MnSO
40.3g/L, NaCl5g/L, K
2hPO
40.3g/L, calcium carbonate 5g/L.
The condition of fermentation culture is: pH nature; Culture temperature 30 DEG C; Rotating speed 200r/min; Oxygen-supply quantity 2m
3/ h; Liquid amount 30L; Inoculum size 8% (v/v); Incubation time 48h.
At the end of cultivation, its viable count is 8.9 × 10
9cfu/mL.
Example 2
Take subtilis as fermented bacterium, be inoculated in fermention medium after three grades of seed activations and cultivate.
(1) activation culture: peptone 10g/L, beef powder 3g/L, sodium-chlor 5g/L, glucose 1g/L; The condition of activation culture is: shaking table is cultivated; PH value 7.5; Temperature is 30 DEG C; Time 24h.
(2) fermentation culture:
Be inoculated in fermention medium by the subtilis activated and cultivate, fermentation volume is 300L.
Described fermention medium is made up of following raw material: glucose 10g/L, soybean cake powder 15g/L, MnSO
40.3g/L, NaCl5g/L, K
2hPO
40.3g/L, calcium carbonate 5g/L.
The condition of fermentation culture is: pH nature; Culture temperature 30 DEG C; Rotating speed 200r/min; Oxygen-supply quantity 15m
3/ h; Liquid amount 30L; Inoculum size 8% (v/v); Incubation time 48h.
At the end of cultivation, its viable count is 8.2 × 10
9cfu/mL.
In above-mentioned two examples, bacillus living number measuring method is: get 1mL fermented liquid after fermentation ends and join in 9mL stroke-physiological saline solution, take turns doing 10 times of serial dilutions, choose 3 suitable extension rates, drawing 0.1mL diluent is injected in plate count substratum, diluent mixing paved with spreading rod, each extent of dilution connects three flat boards, is inverted, in 37 DEG C of constant incubators, cultivate 24h, choose the culture dish meter colony number of colony number between 30 ~ 300.
Claims (4)
1. a method for carrying out high-density culture on bacillus subtilis, is characterized in that comprising step as follows:
(1) be that fermented bacterium carries out three grades of seed activations with subtilis,
Wherein, the component of activation medium comprises: peptone 10g/L, beef powder 3g/L, sodium-chlor 5g/L, glucose 1g/L;
Three grades of seed activation culture condition are: shaking table cultivation, pH value 7.5, temperature are 30 DEG C, time 24h;
(2) strain inoculation after activation is cultivated in fermention medium,
Wherein, the component of fermention medium comprises: glucose 10g/L, soybean cake powder 15g/L, MnSO
40.3g/L, NaCl5g/L, K
2hPO
40.3g/L, calcium carbonate 5g/L;
Fermentation condition is: pH nature, culture temperature 30 DEG C, mixing speed 200r/min, incubation time 48h, obtain the subtilis of high-density culture.
2. the method for carrying out high-density culture on bacillus subtilis according to claim 1, is characterized in that: in described step (1), the inoculum size of three grades of seed activation cultivations is 8%v/v.
3. the method for carrying out high-density culture on bacillus subtilis according to claim 1, is characterized in that: in described step (2), the inoculum size of fermentation culture is 8%v/v.
4. the method for carrying out high-density culture on bacillus subtilis according to claim 1, is characterized in that: in described step (2) during fermentation culture, and the oxygen-supply quantity of the 10L volume that often ferments is greater than 0.5m
3/ h.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635890A (en) * | 2016-11-23 | 2017-05-10 | 山东省农业科学院农业资源与环境研究所 | Method for continuously culturing bacillus subtilis and special fermenting system |
| CN107815431A (en) * | 2017-11-22 | 2018-03-20 | 山东京青农业科技有限公司 | A kind of method with water-soluble raw material culture bacillus subtilis |
| CN110205274A (en) * | 2019-06-12 | 2019-09-06 | 南京巨鲨显示科技有限公司 | A kind of bacillus stearothermophilus gemma generation culture medium |
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| CN102433283A (en) * | 2011-12-19 | 2012-05-02 | 湖南省微生物研究所 | High-density production process for forage bacillus subtilis, microbial inoculum prepared by using forage bacillus subtilis and application of microbial inoculum |
| CN101935624B (en) * | 2010-04-09 | 2012-05-30 | 武汉科诺生物科技股份有限公司 | Bacillus subtillis and method for preparing raw powder of each gram of bacillus subtillis with 1 trillion live germs |
| CN102168045B (en) * | 2010-12-24 | 2013-10-16 | 北京科为博生物科技有限公司 | Bacillus subtilis preparation and preparation method thereof |
| CN103497920A (en) * | 2013-10-17 | 2014-01-08 | 北京沃土天地生物科技有限公司 | Bacillus agent for preventing and curing blight, and manufacturing method and application for bacillus agent |
| CN103773722A (en) * | 2014-01-16 | 2014-05-07 | 中国药科大学 | Salt-tolerance bacillus subtilis with low-temperature biological deamination function and application of bacillus subtilis |
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2014
- 2014-10-23 CN CN201410567431.6A patent/CN104293721A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101935624B (en) * | 2010-04-09 | 2012-05-30 | 武汉科诺生物科技股份有限公司 | Bacillus subtillis and method for preparing raw powder of each gram of bacillus subtillis with 1 trillion live germs |
| CN102168045B (en) * | 2010-12-24 | 2013-10-16 | 北京科为博生物科技有限公司 | Bacillus subtilis preparation and preparation method thereof |
| CN102433283A (en) * | 2011-12-19 | 2012-05-02 | 湖南省微生物研究所 | High-density production process for forage bacillus subtilis, microbial inoculum prepared by using forage bacillus subtilis and application of microbial inoculum |
| CN103497920A (en) * | 2013-10-17 | 2014-01-08 | 北京沃土天地生物科技有限公司 | Bacillus agent for preventing and curing blight, and manufacturing method and application for bacillus agent |
| CN103773722A (en) * | 2014-01-16 | 2014-05-07 | 中国药科大学 | Salt-tolerance bacillus subtilis with low-temperature biological deamination function and application of bacillus subtilis |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635890A (en) * | 2016-11-23 | 2017-05-10 | 山东省农业科学院农业资源与环境研究所 | Method for continuously culturing bacillus subtilis and special fermenting system |
| CN106635890B (en) * | 2016-11-23 | 2019-11-12 | 山东省农业科学院农业资源与环境研究所 | A kind of continuous method for cultivating bacillus subtilis and dedicated fermentation system |
| CN107815431A (en) * | 2017-11-22 | 2018-03-20 | 山东京青农业科技有限公司 | A kind of method with water-soluble raw material culture bacillus subtilis |
| CN110205274A (en) * | 2019-06-12 | 2019-09-06 | 南京巨鲨显示科技有限公司 | A kind of bacillus stearothermophilus gemma generation culture medium |
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