CN103146634A - 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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- CN103146634A CN103146634A CN2013100790710A CN201310079071A CN103146634A CN 103146634 A CN103146634 A CN 103146634A CN 2013100790710 A CN2013100790710 A CN 2013100790710A CN 201310079071 A CN201310079071 A CN 201310079071A CN 103146634 A CN103146634 A CN 103146634A
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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 the microbial pesticide field, be specifically related to magnesium chloride in the application that improves on the bacillus subtilis spore thermotolerance; And utilize magnesium chloride to improve the stable on heating method of bacillus subtilis spore; Relate in addition strong bacillus subtilis microbial agent of a kind of spore heat resistance and preparation method thereof.
Background technology
Subtilis (Bacillus subtilis) is a class important Biocontrol microorganism colony in soil, can prevent and treat plurality of plant diseases (He Liyuan. biological control circular, 1985,1 (3): 28-31; Yu G Y etc., Soil Biology and Biochemistry.2002,34:955-963).Gemma is the stronger hypopus (Shen Ping etc. of a class resistance that genus bacillus produced in the fermentation later stage, microbiology, the .2006 of Higher Education Publishing House, 55-60), often be used as effective constituent and be made into microbial pesticide, but with respect to chemical pesticide, the microbial pesticide shelf-lives take gemma as effective constituent is relatively short, and the temperature capacity of gemma is one of principal element that affects its shelf-lives.
The resistance toheat of gemma is except the control that is subjected to 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+The resistance toheat of gemma there are in various degree raising effect (the .Applied and Environmental Microbiology.2011 such as Granger A C, 77 (1): 32-40).Therefore, the thermotolerance of raising bacillus subtilis spore is one of effective way that extends corresponding microbial pesticide shelf-lives.
Summary of the invention
For the short problem of subtilis microbial pesticide shelf-lives, the object of the invention is to provide magnesium chloride in the application that improves on the bacillus subtilis spore thermotolerance.
Another purpose of the present invention is to provide utilizes magnesium chloride to improve the stable on heating method of bacillus subtilis spore.
The bacillus subtilis microbial agent that provides a kind of spore heat resistance strong is provided the present invention's the 3rd purpose.
The present invention's the 4th purpose is to provide the preparation method of the strong bacillus subtilis microbial agent of above-mentioned spore heat resistance.
Purpose of the present invention realizes by following technical scheme:
Magnesium chloride is in the application that improves on the bacillus subtilis spore thermotolerance.
The present invention utilizes magnesium chloride to improve the stable on heating method of bacillus subtilis spore, comprises in the fermented liquid after fermention medium before cultivating to fermentation of bacillus subtilis or fermentation culture finish adding MgCl
2, stir.
The above-mentioned magnesium chloride that utilizes improves the stable on heating method of bacillus subtilis spore, according to adding 0.1g～0.3g MgCl in every 100mL fermention medium
2Ratio add MgCl in fermention medium
2The 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 magnesium chloride that utilizes improves the stable on heating method of bacillus subtilis spore, according to adding 0.1g～0.3g MgCl in every 100mL fermented liquid
2Ratio add MgCl in fermented liquid
2In 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 of gemma as microbial pesticide effective constituent; July 6 be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center as BAB-1(2007, deposit number is: CGMCC No.2099).
The above-mentioned magnesium chloride that utilizes improves in the stable on heating method of bacillus subtilis spore, and its described fermented liquid is prepared as follows:
(1) actication of culture
The Bacillus subtilis strain of cryopreservation is rule on LB substratum solid plate, then cultivated 20～24 hours under 28 ℃～32 ℃, get activated spawn;
(2) seed liquor preparation
According to the ratio that a transfering loop activated spawn is inoculated in 100mL seed liquor substratum, activated spawn is inoculated in the seed liquor substratum, shaking culture 10～15h under 30 ℃～32 ℃, 170～210r/min condition gets seed liquor;
(3) fermented liquid preparation
Being 3%～5% ratio according to volume percent is inoculated into the seed liquor of step (2) gained in fermention medium, and shaking culture 40～50h under 30 ℃～32 ℃, pH7.0～7.5,170～210r/min condition gets fermented liquid.
Above-mentionedly utilize magnesium chloride to improve LB substratum, seed liquor substratum, the fermention medium described in the stable on heating method of bacillus subtilis spore to prepare according to ordinary method.
Above-mentioned moiety and the mass percent of utilizing magnesium chloride to improve the LB substratum described in the stable on heating method of bacillus subtilis spore is: yeast extract paste 0.5%, and Tryptones 1%, sodium-chlor 0.5%, agar 1.2%, all the other are water.
Above-mentioned moiety and the mass percent of utilizing magnesium chloride to improve the seed liquor substratum described in the stable on heating method of bacillus subtilis spore is: glucose 0.5%, and yeast extract paste 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are water.
Above-mentioned moiety and the mass percent of utilizing magnesium chloride to improve the fermention medium described in the stable on heating method of bacillus subtilis spore 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 finish
2, stir.
Above-mentioned bacillus subtilis microbial agent is according to adding 0.1g～0.3gMgCl in every 100mL fermention medium
2Ratio add MgCl in fermention medium
2The 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 is according to adding 0.1g～0.3g MgCl in every 100mL fermented liquid
2Ratio add MgCl in fermented liquid
2In 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 of gemma as microbial pesticide effective constituent; July 6 be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center as BAB-1(2007, 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 cultivated under 28～32 ℃ 20～24 hours, get activated spawn;
(2) seed liquor preparation
The ratio that is inoculated in 100mL seed liquor substratum according to a transfering loop activated spawn is inoculated in activated spawn in the seed liquor substratum, and shaking culture 10～15h under 30 ℃～32 ℃, 170～210r/min condition gets seed liquor;
(3) fermented liquid preparation
Being 3%～5% ratio according to volume percent is inoculated into the seed liquor of step (2) gained in fermention medium, and shaking culture 40～50h under 30 ℃～32 ℃, pH7.0～7.5,170～210r/min condition gets fermented liquid.
Described subtilis can be that BAB-1(2007 July 6 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is: CGMCC No.2099).
LB substratum described in above-mentioned bacillus subtilis microbial agent, seed liquor substratum, fermention medium prepare according to ordinary method.
Moiety and the mass percent of the LB substratum described in above-mentioned bacillus subtilis microbial agent are: yeast extract paste 0.5%, and Tryptones 1%, sodium-chlor 0.5%, agar 1.2%, 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%, and yeast extract paste 0.5%, peptone 1%, sodium-chlor 0.5%, 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 fermention medium before cultivating to fermentation of bacillus subtilis or fermentation culture finish and adds MgCl
2, stir.
The preparation method of the bacillus subtilis microbial agent that above-mentioned spore heat resistance is strong is according to adding 0.1g～0.3g MgCl in every 100mL fermention medium
2Ratio add MgCl in fermention medium
2The 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 is according to adding 0.1g～0.3g MgCl in every 100mL fermented liquid
2Ratio add MgCl in fermented liquid
2In 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: the Bacillus subtilis strain of cryopreservation is rule in LB substratum solid plate, then cultivated 20～24 hours under 28 ℃～32 ℃, get activated spawn;
(2) seed liquor preparation: be inoculated in the seed liquor substratum according to the bacterial classification of the ratio that a transfering loop activated spawn is inoculated in 100mL seed liquor substratum with activation, shaking culture 10～15h under 30 ℃～32 ℃, 170～210r/min condition gets seed liquor;
(3) fermented liquid preparation: being 3%～5% ratio according to volume percent is inoculated into the seed liquor of step (2) gained in fermention medium, and shaking culture 40～50h under 30 ℃～32 ℃, pH7.0～7.5,170～210r/min condition gets fermented liquid.
LB substratum described in above-mentioned preparation method, seed liquor substratum, fermention medium prepare according to ordinary method.
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: compare with existing bacillus subtilis microbial agent, the present invention is 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.
Description of drawings
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
Below the present invention is further illustrated with specific embodiment, but be construed as limiting the invention never in any form.
Embodiment 1 strengthens the stable on heating metal ion shaker test of bacillus subtilis spore
The subtilis BAB-1 bacterial classification that (1) will be kept at-80 ℃ of refrigerators (was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 6th, 2007, deposit number is: CGMCCNo.2099) rule in LB substratum solid plate, flat board is placed in 30 ℃ of incubator activation culture 24h, gets activated spawn.
(2) (moiety of seed liquor substratum and weight ratio thereof are: glucose 5g to be inoculated in 100mL seed liquor substratum with transfering loop picking one ring activated spawn, yeast extract paste 5g, peptone 10g, sodium-chlor 5g, water 1000mL) in, shaking culture 12h under 31 ℃, 180r/min condition obtains seed liquor.
(3) be that 4% ratio is inoculated into fermention medium with seed liquor (moiety of fermention medium and weight ratio thereof are: glucose 20g, Zulkovsky starch 40g, peptone 1g, NaNO according to volume percent
310g, K
2HPO
43g, MgSO
47H
2O1g, CaCO
31g, water 1000mL) in, shaking culture 48h under 31 ℃, 180r/min condition gets fermented liquid.
(4) sampling 10mL, do gramstaining, examines under a microscope the sporulation situation, greater than 90% the time, stops fermentation at spore forming rate;
(5) get the fermented liquid of 5 parts of equal volume, add respectively metal ion: NaCl, CaCl according to the ratio of 0.2g/100mL in fermented liquid
2, MgCl
2, MnSO
4, FeSO
4, with the processing of not adding any metal ion (CK) in contrast.
(6) will manage fermented liquid everywhere and be placed in 54 ℃ of standing 14d of incubator.
(7) will manage fermented liquid everywhere and take out, get respectively the 1mL sample, dilute with 10 times of concentration gradients with sterilized water, fermented liquid will be diluted to 10
-nThe LB culture dish is divided into four districts, every district's point 20 μ L diluents.Culture dish is placed in 35 ℃ and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculates the quantity of gemma in every mL fermented liquid, relatively the fermented liquid gemma amount of survival of different metal ion processing
Gemma quantity=(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) the gemma quantity that contrasts after 54 ℃ of processing 7d of result (seeing Table 1) is 6.1 * 10
9, NaCl, CaCl
2, MnSO
4And FeSO
4The gemma quantity of processing 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 gemma quantity of processing is 8.4 * 10
9, be significantly higher than the gemma quantity that contrasts with other metal ion treatments, NaCl is described, CaCl
2, MnSO
4And FeSO
4The bacillus subtilis spore thermotolerance is not had impact substantially, and MgCl
2Can significantly improve the temperature capacity of bacillus subtilis spore.
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 agents of the present invention and spore heat resistance simultaneous test
Specifically comprise the steps:
(1), the subtilis BAB-1 bacterial classification that will be kept at-80 ℃ of refrigerators rules in LB substratum solid plate, and flat board is placed in 30 ℃ of incubator activation culture 24h.
(2), (moiety of seed liquor substratum and weight ratio thereof are: glucose 5g to be inoculated in the seed liquor substratum with transfering loop picking one ring activated spawn, yeast extract paste 5g, peptone 10g, sodium-chlor 5g, water 1000mL) in 100ml, shaking culture 12h under 31 ℃, 180r/min condition obtains seed liquor.
(3), be that 4% ratio is inoculated into fermention medium with seed liquor (moiety of fermention medium and weight ratio thereof are: glucose 20g, Zulkovsky starch 40g, peptone 1g, NaNO according to volume ratio
310g, K
2HPO
43g, MgSO
47H
2O1g, CaCO
31g, water 1000mL) in, shaking culture 48h under 31 ℃, 180r/min condition.
(4), sampling 10mL, do gramstaining, examine under a microscope the sporulation situation, stop fermentation greater than 90% the time at spore forming rate.
(5), fermented liquid taken out be divided into 2 parts, add MgCl according to the 0.2g/100mL ratio in a copy of it
2, stir, get microbial inoculum of the present invention; Another part do not process.
(6), with two portions of fermented liquids in (5) 90 ℃ and 100 ℃ of water bath processing 30 minutes, take out and be placed in cold water and be cooled to rapidly room temperature, dilute with 10 times of concentration gradients with sterilized water, fermented liquid is diluted to 10
-nThe LB culture dish is divided into four districts, every district's point 20 μ L diluents.Culture dish is placed in 35 ℃ and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculates the quantity of gemma in every mL fermented liquid, relatively the gemma amount of survival of two portions of fermented liquids
Gemma quantity=(A1+A2+A3+A4)/4 * 10
n* 50(unit: individual/mL).
Annotate: A1, A2, A3, A4 are the colony number in each district of culture dish, and n is extension rate.
Result (seeing Table 2), under 90 ℃ of processing, MgCl
2Processing gemma temperature capacity contrasts and is significantly increased; Under 100 ℃ of processing, MgCl
2Processing gemma survival rate is 7.5 times of control group.MgCl is added in explanation in fermented liquid
2After, the temperature capacity of bacillus subtilis spore significantly improves.
Table 2 MgCl
2Comparative test result on the impact of bacillus subtilis spore temperature capacity
The preparation of embodiment 3 bacillus subtilis microbial agents of the present invention and spore heat resistance simultaneous test
Carry out as follows:
(1) with embodiment 2 steps (1).
(2) with embodiment 2 steps (2).
(3) 2 parts of fermention mediums of preparation, 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 that adds 0.2% on the basis of above-mentioned same composition fermention medium
2
Be that 4% ratio is inoculated into respectively in above-mentioned fermention medium with the seed liquor of step (2) according to volume ratio, shaking culture 48h under 31 ℃, 180r/min condition.
(4) sampling 10mL, do gramstaining, examines under a microscope the sporulation situation, stops fermentation greater than 90% the time at spore forming rate, gets fermented liquid, wherein adds MgCl in fermention medium
2The gained fermented liquid be the suspension of the uninteresting genus bacillus of the present invention.
(5) with two kinds of fermented liquids respectively at 90 ℃ and 100 ℃ of water bath processing 30min, take out and to be placed in cold water and to be cooled to rapidly room temperature, use sterilized water to dilute with 10 times of concentration gradients, fermented liquid is diluted to 10
-nThe LB culture dish is divided into four districts, every district's point 20 μ L diluents.Culture dish is placed in 35 ℃ and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculates the quantity of gemma in every mL fermented liquid, relatively the gemma amount of survival of two portions of fermented liquids
Gemma quantity=(A1+A2+A3+A4)/4 * 10
n* 50(unit: individual/mL).
Annotate: A1, A2, A3, A4 are the colony number in each district of culture dish, and n is extension rate.
Under result (seeing Table 3), 90 ℃ and 100 ℃ of processing, add MgCl
2Fermention medium in the gemma survival rate than not adding MgCl
2Processing all be significantly increased, illustrate in fermention medium to add also can significantly improve the thermotolerance of bacillus subtilis spore.
Table 3 MgCl
2Comparative test result on the impact of bacillus subtilis spore temperature capacity
The shelf-lives length simultaneous test of embodiment 4 bacillus subtilis microbial agents 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 the contrast.
(2) will add MgCl
2Microbial inoculum and do not add MgCl
2The contrast microbial inoculum be placed in 45 ℃ of placements of same incubator, carry out every other month sample thief 1mL.
(3) sample is diluted with 10 times of concentration gradients with sterilized water, fermented liquid is diluted to 10
-nThe LB culture dish is divided into four districts, every district's point 20 μ L diluents.Culture dish is placed in 35 ℃ and cultivates 8h, counts every district bacterium colony, is advisable with 10-40 bacterium colony concentration, and calculates the quantity of gemma in every mL microbial inoculum, relatively the gemma amount of survival of two portions of microbial inoculums
Gemma quantity=(A1+A2+A3+A4)/4 * 10
n* 50(unit: individual/mL).
Annotate: A1, A2, A3, A4 are the colony number in each district of culture dish, and n is extension rate.
(4) to adding MgCl in fermented liquid according to making in embodiment 2
2Microbial inoculum carry out the detection of 12 months, (see figure 1) is as a result added MgCl
2The gemma content of bacillus subtilis microbial agent reduce gradually in the monitoring of 12 months, the reduction degree is slow, finally gemma content is 4.05 * 10 in the time of 12 months
9Individual/mL, and do not add MgCl
2Processing in, gemma content descends rapidly, gemma content is only 0.8 * 10 in the time of 12 months
8Individual/mL.MgCl is added in explanation in fermented liquid
2The bacillus subtilis microbial agent shelf-lives of gained is long.
(5) to adding MgCl in fermention medium according to making in embodiment 3
2Microbial inoculum carry out the detection of 12 months, (see figure 2) is added MgCl as a result
2The gemma content of bacillus subtilis microbial agent reduce gradually in the monitoring of 12 months, the reduction degree is slow, finally gemma content is 7.2 * 10 in the time of 12 months
8Individual/mL, and do not add MgCl
2Processing in, gemma content descends rapidly, gemma content is only 0.4 * 10 in the time of 12 months
8Individual/mL.Show and add MgCl in fermention medium
2The bacillus subtilis microbial agent shelf-lives of gained is long.
Claims (10)
1. magnesium chloride is in the application that improves on the bacillus subtilis spore thermotolerance.
2. utilize magnesium chloride to improve the stable on heating method of bacillus subtilis spore, add MgCl in the fermented liquid after the fermention medium before it is characterized in that cultivating to fermentation of bacillus subtilis or fermentation culture finish
2, stir.
3. it is characterized in that according to adding 0.1g～0.3g MgCl in every 100mL fermention medium in accordance with the method for claim 2,
2Ratio add MgCl in fermention medium
2The 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.
4. it is characterized in that according to adding 0.1g～0.3g MgCl in every 100mL fermented liquid in accordance with the method for claim 2,
2Ratio add MgCl in fermented liquid
2In 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.
5. according to the described method of claim 2 or 4, it is characterized in that described fermented liquid is prepared as follows:
(1), the Bacillus subtilis strain of cryopreservation is rule on LB substratum solid plate, then cultivated 20～24 hours under 28 ℃～32 ℃, get activated spawn;
(2), according to the ratio that a transfering loop activated spawn is inoculated in 100mL seed liquor substratum, activated spawn is inoculated in the seed liquor substratum, shaking culture 10～15h under 30 ℃～32 ℃, 170～210r/min condition gets seed liquor;
(3), to be 3%～5% ratio according to volume percent be inoculated into the seed liquor of step (2) gained in fermention medium, shaking culture 40～50h under 30 ℃～32 ℃, pH7.0～7.5,170～210r/min condition gets fermented liquid.
6. in accordance with the method for claim 5, 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; The moiety of described seed liquor substratum and mass percent are: glucose 0.5%, and yeast extract paste 0.5%, peptone 1%, sodium-chlor 0.5%, all the other are water; The moiety of described 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.
7. 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 finish
2, stir.
8. according to bacillus subtilis microbial agent claimed in claim 7, it is characterized in that according to adding 0.1g～0.3g MgCl in every 100mL fermention medium
2Ratio add MgCl in fermention medium
2The 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.
9. according to bacillus subtilis microbial agent claimed in claim 7, it is characterized in that according to adding 0.1g～0.3g MgCl in every 100mL fermented liquid
2Ratio add MgCl in fermented liquid
2In 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.
10. according to bacillus subtilis microbial agent claimed in claim 7, it is characterized in that described fermented liquid, be prepared as follows:
(1), the Bacillus subtilis strain of cryopreservation is rule in LB substratum solid plate, then cultivated under 28～32 ℃ 20～24 hours, get activated spawn;
(2), the ratio that is inoculated in 100mL seed liquor substratum according to a transfering loop activated spawn is inoculated in activated spawn in the seed liquor substratum, shaking culture 10～15h under 30 ℃～32 ℃, 170～210r/min condition gets seed liquor;
(3), to be 3%～5% ratio according to volume percent be inoculated into the seed liquor of step (2) gained in fermention medium, shaking culture 40～50h under 30 ℃～32 ℃, pH7.0～7.5,170～210r/min condition gets fermented liquid.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310079071.0A CN103146634B (en) | 2013-03-13 | 2013-03-13 | Application of magnesium chloride in improving spore heat resistance of bacillus subtilis |
Applications Claiming Priority (1)
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|---|---|---|---|
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| 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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| 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 |
Non-Patent Citations (5)
| Title |
|---|
| A.E. CAZEMIER ET AL: "Effect of sporulation and recovery medium on the heat resistance and amount of injury of spores from spoilage bacilli", 《JOURNAL OF APPLIED MICROBIOLOGY》, 31 December 2001 (2001-12-31), pages 761 - 770 * |
| F. F. BUSTA ET AL: "Heat-induced requirements for sucrose or magnesium for expression of heat resistance in Bacillus cereus forespores", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》, vol. 32, no. 2, 31 August 1976 (1976-08-31), pages 312 - 314 * |
| O. B. WILLIAMS: "The heat resistance of Bacterial Spores", 《THE JOURNAL OF INFECTIOUS DISEASES》, vol. 44, no. 6, 30 June 1929 (1929-06-30), pages 421 - 465 * |
| 姚露燕 等: "金属离子浓度对枯草芽孢杆菌芽孢率的影响", 《现代食品科技》, vol. 24, no. 8, 31 December 2008 (2008-12-31), pages 770 - 772 * |
| 张晓云: "枯草芽孢杆菌菌株CAB-1抑菌物质的分离鉴定及活性分析", 《中国优秀硕士学位论文全文数据库农业科技辑》, 15 July 2011 (2011-07-15), pages 046 - 77 * |
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