CN103146634B - Application of magnesium chloride in improving spore heat resistance of bacillus subtilis - Google Patents
Application of magnesium chloride in improving spore heat resistance of bacillus subtilis Download PDFInfo
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Abstract
The invention discloses application of magnesium chloride in improving spore heat resistance of bacillus subtilis and belongs to the field of microbial pesticides. The invention further discloses a method for improving the spore heat resistance of bacillus subtilis by utilizing magnesium chloride as well as a bacillus subtilis agent with strong spore heat resistance. The bacillus subtilis agent with strong spore heat resistance is obtained by adding MgCl2 to a fermentation medium before fermentation culture of bacillus subtilis or a fermentation liquor after fermentation culture of bacillus subtilis, wherein 0.1g-0.3g of MgCl2 is added to every 100 mL of fermentation medium or fermentation liquor. In the bacillus subtilis agent obtained by adding MgCl2, the spore heat resistance is remarkably improved; and, the shelf life of the bacillus subtilis agent disclosed by the invention is long.
Description
Technical field
The invention belongs to microbial pesticide field, be specifically related to magnesium chloride and improving the application in bacillus subtilis spore thermotolerance; And utilize magnesium chloride to improve the method for bacillus subtilis spore thermotolerance; Relate to strong bacillus subtilis microbial agent of a kind of spore heat resistance and preparation method thereof in addition.
Background technology
Subtilis (Bacillus subtilis) is the Biocontrol microorganism colony that in soil, a class is important, can prevent and treat plurality of plant diseases (He Liyuan. biological control circulate a notice of, 1985,1 (3): 28-31; Yu G Y etc., Soil Biology and Biochemistry.2002,34:955-963).Gemma is the genus bacillus stronger hypopus (Shen Ping etc. of a class resistance that produce of phase after fermentation, microbiology, Higher Education Publishing House .2006,55-60), often be made into microbial pesticide by as effective constituent, but relative to chemical pesticide, be that the microbial pesticide shelf-lives of effective constituent is relatively short with gemma, and the temperature capacity of gemma is one of principal element affecting its shelf-lives.
The resistance toheat of gemma is except being subject to the control of genes involved, and in external environment, metal ion, temperature, potential of hydrogen etc. all can affect formation and the performance of gemma, wherein metallic ions Ca
2+, Mn
2+, Fe
2+raising effect (the .Applied and Environmental Microbiology.2011 such as Granger A C, 77 (1): 32-40) is in various degree had to the resistance toheat of gemma.Therefore, the thermotolerance improving bacillus subtilis spore is one of effective way extending corresponding microbial pesticide shelf-lives.
Summary of the invention
For the problem that microorganism Bacillus subtilis agricultural chemicals shelf-lives is short, the object of the invention is to provide magnesium chloride improving the application in bacillus subtilis spore thermotolerance.
Another object of the present invention is to provide the method utilizing magnesium chloride to improve bacillus subtilis spore thermotolerance.
The present invention the 3rd object is the bacillus subtilis microbial agent providing a kind of spore heat resistance strong.
The present invention the 4th object is to provide the preparation method of the bacillus subtilis microbial agent that above-mentioned spore heat resistance is strong.
Object of the present invention is realized by following technical scheme:
Magnesium chloride is improving the application in bacillus subtilis spore thermotolerance.
The present invention utilizes magnesium chloride to improve the method for bacillus subtilis spore thermotolerance, comprises in the fermented liquid after terminating to the fermention medium before fermentation of bacillus subtilis cultivation or fermentation culture and adds MgCl
2, stir.
The above-mentioned method utilizing magnesium chloride to improve bacillus subtilis spore thermotolerance, adds 0.1g ~ 0.3g MgCl according in every 100mL fermention medium
2ratio in fermention medium, add MgCl
2; The moiety of wherein said fermention medium and mass percent are: glucose 2% ~ 2.5%, Zulkovsky starch 3% ~ 5%, peptone 0.1% ~ 0.2%, NaNO
31% ~ 2%, K
2hPO
40.2% ~ 0.4%, MgSO
47H
2o0.1% ~ 0.2%, CaCO
30.05% ~ 0.15%, all the other are water.
The above-mentioned method utilizing magnesium chloride to improve bacillus subtilis spore thermotolerance, adds 0.1g ~ 0.3g MgCl according in every 100mL fermented liquid
2ratio in fermented liquid, add MgCl
2; In wherein said fermented liquid, the total count of subtilis is 1.0 × 10
9~ 1.0 × 10
10individual/mL, wherein gemma accounts for 90% of subtilis total count.
Described subtilis refers to the subtilis using gemma as microbial pesticide effective constituent; As BAB-1(2007 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center July 6, deposit number is: CGMCC No.2099).
The above-mentioned magnesium chloride that utilizes improves in the method for bacillus subtilis spore thermotolerance, and the fermented liquid described in it is prepared as follows:
(1) actication of culture
The Bacillus subtilis strain of cryopreservation is rule on LB substratum solid plate, then cultivates 20 ~ 24 hours at 28 DEG C ~ 32 DEG C, obtain activated spawn;
(2) seed liquor preparation
According to a ratio transfering loop activated spawn being inoculated in 100mL seed liquor substratum, activated spawn is inoculated in seed liquor substratum, at 30 DEG C ~ 32 DEG C, shaking culture 10 ~ 15h under 170 ~ 210r/min condition, obtains seed liquor;
(3) fermented liquid preparation
According to volume percent be 3% ~ 5% ratio the seed liquor of step (2) gained is inoculated in fermention medium, 30 DEG C ~ 32 DEG C, pH7.0 ~ 7.5, shaking culture 40 ~ 50h under 170 ~ 210r/min condition, obtain fermented liquid.
The above-mentioned LB substratum described in method of magnesium chloride raising bacillus subtilis spore thermotolerance, seed liquor substratum, the fermention medium of utilizing conventionally is prepared.
The above-mentioned moiety of the LB substratum described in method of magnesium chloride raising bacillus subtilis spore thermotolerance and the mass percent of utilizing is: yeast extract paste 0.5%, Tryptones 1%, sodium-chlor 0.5%, agar 1.2%, all the other are water.
The above-mentioned moiety of the seed liquor substratum described in method of magnesium chloride raising bacillus subtilis spore thermotolerance and the mass percent of utilizing is: glucose 0.5%, yeast extract paste 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are water.
The above-mentioned moiety of the fermention medium described in method of magnesium chloride raising bacillus subtilis spore thermotolerance and the mass percent of utilizing is: glucose 2% ~ 2.5%, Zulkovsky starch 3% ~ 5%, peptone 0.1% ~ 0.2%, NaNO
31% ~ 2%, K
2hPO
40.2% ~ 0.4%, MgSO
47H
2o0.1% ~ 0.2%, CaCO
30.05% ~ 0.15%, all the other are water.
The bacillus subtilis microbial agent that a kind of spore heat resistance of the present invention is strong, is prepared as follows: add MgCl in the fermented liquid after the fermention medium before cultivating to fermentation of bacillus subtilis or fermentation culture terminate
2, stir.
Above-mentioned bacillus subtilis microbial agent, adds 0.1g ~ 0.3gMgCl according in every 100mL fermention medium
2ratio in fermention medium, add MgCl
2; The moiety of wherein said fermention medium and mass percent are: glucose 2% ~ 2.5%, Zulkovsky starch 3% ~ 5%, peptone 0.1% ~ 0.2%, NaNO
31% ~ 2%, K
2hPO
40.2% ~ 0.4%, MgSO
47H
2o0.1% ~ 0.2%, CaCO
30.05% ~ 0.15%, all the other are water.
Above-mentioned bacillus subtilis microbial agent, adds 0.1g ~ 0.3g MgCl according in every 100mL fermented liquid
2ratio in fermented liquid, add MgCl
2; In wherein said fermented liquid, the total count of subtilis is 1.0 × 10
9~ 1.0 × 10
10individual/mL, wherein gemma accounts for 90% of subtilis total count.
Described subtilis refers to the subtilis using gemma as microbial pesticide effective constituent; As BAB-1(2007 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center July 6, deposit number is: CGMCC No.2099).
Fermented liquid described in above-mentioned bacillus subtilis microbial agent, is prepared as follows:
(1) actication of culture
The Bacillus subtilis strain of cryopreservation is rule in LB substratum solid plate, then cultivates 20 ~ 24 hours at 28 ~ 32 DEG C, obtain activated spawn;
(2) seed liquor preparation
Activated spawn is inoculated in seed liquor substratum by the ratio being inoculated in 100mL seed liquor substratum according to a transfering loop activated spawn, at 30 DEG C ~ 32 DEG C, shaking culture 10 ~ 15h under 170 ~ 210r/min condition, obtains seed liquor;
(3) fermented liquid preparation
According to volume percent be 3% ~ 5% ratio the seed liquor of step (2) gained is inoculated in fermention medium, 30 DEG C ~ 32 DEG C, pH7.0 ~ 7.5, shaking culture 40 ~ 50h under 170 ~ 210r/min condition, obtain fermented liquid.
Described subtilis can be that BAB-1(2007 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center July 6, and deposit number is: CGMCC No.2099).
LB substratum described in above-mentioned bacillus subtilis microbial agent, seed liquor substratum, fermention medium are conventionally prepared.
Moiety and the mass percent of the LB substratum described in above-mentioned bacillus subtilis microbial agent are: yeast extract paste 0.5%, Tryptones 1%, sodium-chlor 0.5%, agar 1.2%, and all the other are water.
Moiety and the mass percent of the seed liquor substratum described in above-mentioned bacillus subtilis microbial agent are: glucose 0.5%, yeast extract paste 0.5%, peptone 1%, sodium-chlor 0.5%, and all the other are water.
The moiety of the fermention medium described in above-mentioned bacillus subtilis microbial agent and mass percent are: glucose 2% ~ 2.5%, Zulkovsky starch 3% ~ 5%, peptone 0.1% ~ 0.2%, NaNO
31% ~ 2%, K
2hPO
40.2% ~ 0.4%, MgSO
47H
2o0.1% ~ 0.2%, CaCO
30.05% ~ 0.15%, all the other are water.
The preparation method of the bacillus subtilis microbial agent that above-mentioned spore heat resistance is strong, comprises in the fermented liquid after terminating to the fermention medium before fermentation of bacillus subtilis cultivation or fermentation culture and adds MgCl
2, stir.
The preparation method of the bacillus subtilis microbial agent that above-mentioned spore heat resistance is strong, adds 0.1g ~ 0.3g MgCl according in every 100mL fermention medium
2ratio in fermention medium, add MgCl
2; The moiety of wherein said fermention medium and mass percent are: glucose 2% ~ 2.5%, Zulkovsky starch 3 ~ 5%, peptone 0.1% ~ 0.2%, NaNO
31% ~ 2%, K
2hPO
40.2% ~ 0.4%, MgSO
47H
2o0.1% ~ 0.2%, CaCO
30.05% ~ 0.15%, all the other are water.
The preparation method of the bacillus subtilis microbial agent that above-mentioned spore heat resistance is strong, adds 0.1g ~ 0.3g MgCl according in every 100mL fermented liquid
2ratio in fermented liquid, add MgCl
2; In wherein said fermented liquid, the total count of subtilis is 1.0 × 10
9~ 1.0 × 10
10individual/mL, wherein gemma accounts for 90% of subtilis total count.
Fermentation of bacillus subtilis liquid described in above-mentioned preparation method, is prepared as follows:
(1) actication of culture: rule in LB substratum solid plate by the Bacillus subtilis strain of cryopreservation, then cultivates 20 ~ 24 hours, obtains activated spawn at 28 DEG C ~ 32 DEG C;
(2) seed liquor preparation: according to a ratio transfering loop activated spawn being inoculated in 100mL seed liquor substratum by the strain inoculation of activation in seed liquor substratum, at 30 DEG C ~ 32 DEG C, shaking culture 10 ~ 15h under 170 ~ 210r/min condition, obtain seed liquor;
(3) fermented liquid preparation: according to volume percent be 3% ~ 5% ratio the seed liquor of step (2) gained is inoculated in fermention medium, 30 DEG C ~ 32 DEG C, pH7.0 ~ 7.5, shaking culture 40 ~ 50h under 170 ~ 210r/min condition, obtain fermented liquid.
LB substratum described in above-mentioned preparation method, seed liquor substratum, fermention medium are conventionally prepared.
The bacillus subtilis microbial agent that spore heat resistance of the present invention is strong can be prepared into the pesticide formulation such as suspension agent, wettable powder, effervescent tablet, granule according to mode well known to those skilled in the art.
The advantage that the present invention has with beneficial effect is: compared with existing bacillus subtilis microbial agent, and the present invention by adding MgCl in fermentation culture or fermention medium
2in the bacillus subtilis microbial agent of gained, its gemma temperature capacity significantly improves; Bacillus subtilis microbial agent shelf-lives of the present invention is long.
Accompanying drawing explanation
Fig. 1. add MgCl in fermented liquid
2the shelf-lives graphic representation of bacillus subtilis microbial agent.
Fig. 2. add MgCl in fermention medium
2the shelf-lives graphic representation of bacillus subtilis microbial agent.
Embodiment
With specific embodiment, the present invention is further illustrated below, but be construed as limiting the invention never in any form.
Embodiment 1 strengthens the metal ion shaker test of bacillus subtilis spore thermotolerance
(1) by the subtilis BAB-1 bacterial classification that is kept at-80 DEG C of refrigerators, (on July 6th, 2007 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is: CGMCCNo.2099) rule in LB substratum solid plate, flat board is placed in 30 DEG C of incubator activation culture 24h, obtains activated spawn.
(2) (moiety of seed liquor substratum and weight ratio thereof are: glucose 5g to be inoculated in 100mL seed liquor substratum by transfering loop picking one ring activated spawn, yeast extract paste 5g, peptone 10g, sodium-chlor 5g, water 1000mL) in, at 31 DEG C, shaking culture 12h under 180r/min condition, obtain seed liquor.
(3) according to volume percent be 4% ratio seed liquor is inoculated into fermention medium (moiety of fermention medium and weight ratio thereof is: glucose 20g, Zulkovsky starch 40g, peptone 1g, NaNO
310g, K
2hPO
43g, MgSO
47H
2o1g, CaCO
31g, water 1000mL) in, at 31 DEG C, shaking culture 48h under 180r/min condition, obtain fermented liquid.
(4) sample 10mL, do gramstaining, examine under a microscope sporulation situation, when spore forming rate is greater than 90%, stop fermentation;
(5) get the fermented liquid of 5 parts of same volumes, in fermented liquid, add metal ion according to the ratio of 0.2g/100mL respectively: NaCl, CaCl
2, MgCl
2, MnSO
4, FeSO
4, not add the process of any metal ion (CK) in contrast.
(6) fermented liquid will be managed everywhere and be placed in the standing 14d of 54 DEG C of incubators.
(7) fermented liquid will be managed everywhere take out, and get 1mL sample respectively, dilute with 10 times of concentration gradients with sterilized water, fermented liquid is diluted to 10
-n.LB culture dish is divided into four districts, every district point 20 μ L diluent.Culture dish is placed in 35 DEG C and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculate the quantity of gemma in every mL fermented liquid, compare the fermented liquid gemma amount of survival of different metal ion processing
Number of spores=(A1+A2+A3+A4)/4 × 10
n× 50(unit: individual/mL).
Wherein A1, A2, A3, A4 are the colony number in each district of culture dish, and n is extension rate.
(8) Number of spores contrasted after result (see table 1) 54 DEG C process 7d is 6.1 × 10
9, NaCl, CaCl
2, MnSO
4and FeSO
4the Number of spores of process is respectively 6.0 × 10
9, 5.5 × 10
9, 6.0 × 10
9, 6.2 × 10
9, 6.2 × 10
9, and MgCl
2the Number of spores of process is 8.4 × 10
9, be significantly higher than the Number of spores of contrast and other metal ion treatments, NaCl be described, CaCl
2, MnSO
4and FeSO
4bacillus subtilis spore thermotolerance is not affected substantially, and MgCl
2the temperature capacity of bacillus subtilis spore can be significantly improved.
The test-results of the impact of table 1 different metal ion pair bacillus subtilis spore temperature capacity
The preparation of embodiment 2 bacillus subtilis microbial agent of the present invention and spore heat resistance simultaneous test
Specifically comprise the steps:
(1), by the subtilis BAB-1 bacterial classification being kept at-80 DEG C of refrigerators rule in LB substratum solid plate, flat board is placed in 30 DEG C of incubator activation culture 24h.
(2) (moiety of seed liquor substratum and weight ratio thereof are: glucose 5g, to be inoculated in seed liquor substratum by transfering loop picking one ring activated spawn, yeast extract paste 5g, peptone 10g, sodium-chlor 5g, water 1000mL) in 100ml, at 31 DEG C, shaking culture 12h under 180r/min condition, obtain seed liquor.
(3), according to volume ratio be 4% ratio seed liquor is inoculated into fermention medium (moiety of fermention medium and weight ratio thereof is: glucose 20g, Zulkovsky starch 40g, peptone 1g, NaNO
310g, K
2hPO
43g, MgSO
47H
2o1g, CaCO
31g, water 1000mL) in, at 31 DEG C, shaking culture 48h under 180r/min condition.
(4), sample 10mL, do gramstaining, examine under a microscope sporulation situation, stop fermentation when spore forming rate is greater than 90%.
(5), by fermented liquid take out and be divided into 2 parts, in a copy of it, add MgCl according to 0.2g/100mL ratio
2, stir, obtain microbial inoculum of the present invention; Another part does not process.
(6), by two portions of fermented liquids in (5) 90 DEG C and 100 DEG C of water bath processing 30 minutes, take out and be placed in cold water and be cooled to room temperature rapidly, dilute with 10 times of concentration gradients with sterilized water, fermented liquid is diluted to 10
-n.LB culture dish is divided into four districts, every district point 20 μ L diluent.Culture dish is placed in 35 DEG C and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculate the quantity of gemma in every mL fermented liquid, compare the gemma amount of survival of two portions of fermented liquids
Number of spores=(A1+A2+A3+A4)/4 × 10
n× 50(unit: individual/mL).
Note: A1, A2, A3, A4 are the colony number in each district of culture dish, n is extension rate.
Result (see table 2), under 90 DEG C of process, MgCl
2process gemma temperature capacity comparatively contrasts and is significantly increased; Under 100 DEG C of process, MgCl
2process gemma survival rate is 7.5 times of control group.Illustrate and add MgCl in fermented liquid
2after, the temperature capacity of bacillus subtilis spore significantly improves.
Table 2 MgCl
2on the comparative test result of the impact of bacillus subtilis spore temperature capacity
The preparation of embodiment 3 bacillus subtilis microbial agent of the present invention and spore heat resistance simultaneous test
Carry out as follows:
(1) with embodiment 2 step (1).
(2) with embodiment 2 step (2).
(3) prepare 2 parts of fermention mediums, a copy of it does not add MgCl
2, its moiety and weight ratio thereof are: glucose 20g, Zulkovsky starch 40g, peptone 1g, NaNO
310g, K
2hPO
43g, MgSO
47H
2o1g, CaCO
31g, water 1000mL.Another part adds MgCl
2, namely according to the MgCl adding 0.2% on the basis of above-mentioned same composition fermention medium
2.
The seed liquor of step (2) is inoculated in above-mentioned fermention medium according to the ratio that volume ratio is 4% respectively, at 31 DEG C, shaking culture 48h under 180r/min condition.
(4) sample 10mL, do gramstaining, examine under a microscope sporulation situation, stop fermentation when spore forming rate is greater than 90%, obtain fermented liquid, wherein add MgCl in fermention medium
2gained fermented liquid be the suspension of the uninteresting genus bacillus of the present invention.
(5) by two kinds of fermented liquids respectively at 90 DEG C and 100 DEG C of water bath processing 30min, take out and be placed in cold water and be cooled to room temperature rapidly, use sterilized water to dilute with 10 times of concentration gradients, fermented liquid is diluted to 10
-n.LB culture dish is divided into four districts, every district point 20 μ L diluent.Culture dish is placed in 35 DEG C and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculate the quantity of gemma in every mL fermented liquid, compare the gemma amount of survival of two portions of fermented liquids
Number of spores=(A1+A2+A3+A4)/4 × 10
n× 50(unit: individual/mL).
Note: A1, A2, A3, A4 are the colony number in each district of culture dish, n is extension rate.
Result (see table 3), 90 DEG C and 100 DEG C process under, add MgCl
2fermention medium in gemma survival rate than not adding MgCl
2process be all significantly increased, the thermotolerance of adding in the fermentation medium and also can significantly improve bacillus subtilis spore is described.
Table 3 MgCl
2on the comparative test result of the impact of bacillus subtilis spore temperature capacity
The shelf-lives length simultaneous test of embodiment 4 bacillus subtilis microbial agent of the present invention
Carry out as follows:
(1) according to the method for embodiment 2 and embodiment 3, be prepared into respectively and add 0.2% concentration MgCl in fermention medium or fermented liquid
2bacillus subtilis microbial agent, not add MgCl
2bacillus subtilis microbial agent for contrast.
(2) MgCl will be added
2microbial inoculum and do not add MgCl
2contrast microbial inoculum be placed in same incubator 45 DEG C placement, carry out sample thief 1mL every other month.
(3) sample sterilized water is diluted with 10 times of concentration gradients, fermented liquid is diluted to 10
-n.LB culture dish is divided into four districts, every district point 20 μ L diluent.Culture dish is placed in 35 DEG C and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculate the quantity of gemma in every mL microbial inoculum, compare the gemma amount of survival of two portions of microbial inoculums
Number of spores=(A1+A2+A3+A4)/4 × 10
n× 50(unit: individual/mL).
Note: A1, A2, A3, A4 are the colony number in each district of culture dish, n is extension rate.
(4) MgCl is added to according in fermented liquid obtained in embodiment 2
2microbial inoculum carry out the detection of 12 months, result (see figure 1), add MgCl
2the spore content of bacillus subtilis microbial agent reduce gradually in the monitoring of 12 months, reduce degree slow, finally 12 months time, spore content is 4.05 × 10
9individual/mL, and do not add MgCl
2process in, spore content declines rapidly, and 12 months time, spore content is only 0.8 × 10
8individual/mL.Illustrate and add MgCl in fermented liquid
2the bacillus subtilis microbial agent shelf-lives of gained is long.
(5) MgCl is added to according in fermention medium obtained in embodiment 3
2microbial inoculum carry out the detection of 12 months, result (see figure 2) adds MgCl
2the spore content of bacillus subtilis microbial agent reduce gradually in the monitoring of 12 months, reduce degree slow, finally 12 months time, spore content is 7.2 × 10
8individual/mL, and do not add MgCl
2process in, spore content declines rapidly, and 12 months time, spore content is only 0.4 × 10
8individual/mL.Show to add MgCl in the fermentation medium
2the bacillus subtilis microbial agent shelf-lives of gained is long.
Claims (3)
1. utilize magnesium chloride to improve the method for bacillus subtilis spore thermotolerance, it is characterized in that adding MgCl in the fermented liquid after terminating to the fermention medium before fermentation of bacillus subtilis cultivation or fermentation culture
2, stir; 0.1g ~ 0.3g MgCl is added according in every 100mL fermention medium
2ratio in fermention medium, add MgCl
2; The moiety of wherein said fermention medium and mass percent are: glucose 2% ~ 2.5%, Zulkovsky starch 3% ~ 5%, peptone 0.1% ~ 0.2%, NaNO
31% ~ 2%, K
2hPO
40.2% ~ 0.4%, MgSO
47H
2o0.1% ~ 0.2%, CaCO
30.05% ~ 0.15%, all the other are water; Wherein said subtilis refers to BAB-1, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 6th, 2007, deposit number is: CGMCC No.2099; Or add 0.1g ~ 0.3g MgCl according in every 100mL fermented liquid
2ratio in fermented liquid, add MgCl
2; Described fermented liquid is prepared as follows:
(1), by the Bacillus subtilis strain of cryopreservation rule on LB substratum solid plate, then cultivate 20 ~ 24 hours at 28 DEG C ~ 32 DEG C, obtain activated spawn; Wherein said subtilis refers to BAB-1;
(2), according to a ratio transfering loop activated spawn being inoculated in 100mL seed liquor substratum, activated spawn is inoculated in seed liquor substratum, at 30 DEG C ~ 32 DEG C, shaking culture 10 ~ 15h under 170 ~ 210r/min condition, obtains seed liquor;
(3), according to volume percent be 3% ~ 5% ratio the seed liquor of step (2) gained is inoculated in fermention medium, 30 DEG C ~ 32 DEG C, pH7.0 ~ 7.5, shaking culture 40 ~ 50h under 170 ~ 210r/min condition, obtain fermented liquid.
2. in accordance with the method for claim 1, it is characterized in that the moiety of described LB substratum and mass percent are: yeast extract paste 0.5%, Tryptones 1%, sodium-chlor 0.5%, agar 1.2%, all the other are water; Moiety and the mass percent of described seed liquor substratum are: glucose 0.5%, yeast extract paste 0.5%, peptone 1%, sodium-chlor 0.5%, and all the other are water.
3. the bacillus subtilis microbial agent that spore heat resistance is strong, is characterized in that being prepared as follows: add MgCl in the fermented liquid after the fermention medium before cultivating to fermentation of bacillus subtilis or fermentation culture terminate
2, stir; 0.1g ~ 0.3g MgCl is added according in every 100mL fermention medium
2ratio in fermention medium, add MgCl
2; The moiety of wherein said fermention medium and mass percent are: glucose 2% ~ 2.5%, Zulkovsky starch 3% ~ 5%, peptone 0.1% ~ 0.2%, NaNO
31% ~ 2%, K
2hPO
40.2% ~ 0.4%, MgSO
47H
2o0.1% ~ 0.2%, CaCO
30.05% ~ 0.15%, all the other are water; Wherein said subtilis refers to BAB-1, and be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 6th, 2007, deposit number is: CGMCC No.2099; Or add 0.1g ~ 0.3g MgCl according in every 100mL fermented liquid
2ratio in fermented liquid, add MgCl
2; Described fermented liquid is prepared as follows:
(1), by the Bacillus subtilis strain of cryopreservation rule in LB substratum solid plate, then cultivate 20 ~ 24 hours at 28 ~ 32 DEG C, obtain activated spawn; Wherein said subtilis refers to BAB-1; Moiety and the mass percent of described LB substratum are: yeast extract paste 0.5%, Tryptones 1%, sodium-chlor 0.5%, agar 1.2%, and all the other are water;
(2) activated spawn is inoculated in seed liquor substratum by the ratio, according to a transfering loop activated spawn being inoculated in 100mL seed liquor substratum, at 30 DEG C ~ 32 DEG C, shaking culture 10 ~ 15h under 170 ~ 210r/min condition, obtains seed liquor; Moiety and the mass percent of described seed liquor substratum are: glucose 0.5%, yeast extract paste 0.5%, peptone 1%, sodium-chlor 0.5%, and all the other are water;
(3), according to volume percent be 3% ~ 5% ratio the seed liquor of step (2) gained is inoculated in fermention medium, 30 DEG C ~ 32 DEG C, pH7.0 ~ 7.5, shaking culture 40 ~ 50h under 170 ~ 210r/min condition, obtain fermented liquid.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586142A (en) * | 2012-02-09 | 2012-07-18 | 河北省农林科学院植物保护研究所 | Bacillus subtilis for preventing and curing cucumber downy mildew and microbial inoculants thereof |
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2013
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586142A (en) * | 2012-02-09 | 2012-07-18 | 河北省农林科学院植物保护研究所 | Bacillus subtilis for preventing and curing cucumber downy mildew and microbial inoculants thereof |
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| Title |
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| Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli;A.E. Cazemier et al;《Journal of Applied Microbiology》;20011231;761-770 * |
| Heat-induced requirements for sucrose or magnesium for expression of heat resistance in Bacillus cereus forespores;F. F. BUSTA et al;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19760831;第32卷(第2期);312-314 * |
| The heat resistance of Bacterial Spores;O. B. Williams;《The Journal of Infectious Diseases》;19290630;第44卷(第6期);421-465 * |
| 枯草芽孢杆菌菌株CAB-1抑菌物质的分离鉴定及活性分析;张晓云;《中国优秀硕士学位论文全文数据库农业科技辑》;20110715;D046-77 * |
| 金属离子浓度对枯草芽孢杆菌芽孢率的影响;姚露燕 等;《现代食品科技》;20081231;第24卷(第8期);770-772 * |
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