CN104894032A - Bacillus subtilis growth acceleration and endospore generation culture method - Google Patents

Bacillus subtilis growth acceleration and endospore generation culture method Download PDF

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Publication number
CN104894032A
CN104894032A CN201510345610.XA CN201510345610A CN104894032A CN 104894032 A CN104894032 A CN 104894032A CN 201510345610 A CN201510345610 A CN 201510345610A CN 104894032 A CN104894032 A CN 104894032A
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bacillus subtilis
fermentation
value
glucose
initial
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孟利强
赵晓宇
张淑梅
李晶
曹旭
胡基华
陈镜宇
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Institute of Microbiology of Heilongjiang Academy of Sciences
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Institute of Microbiology of Heilongjiang Academy of Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The invention discloses a bacillus subtilis growth acceleration and endospore generation culture method and aims to solve the problems of low viable count, long fermentation period, low endospore generation rate, difficulty in control of endospore generation related process conditions in a liquid fermentation process of bacillus subtilis. The method includes: activating strains; performing liquid fermentation in shake flasks for seed culture, carrying out fermentation cultivation until residual sugar content of the strains is 0.1%, adding supplementary liquid to control the residual sugar content to range from 0.1 to 0.2, fermenting, supplementing for 5-7h, stopping supplementation and continuing to ferment for 4h, adjusting the pH value to range from 8.5 to 9 and controlling dissolved oxygen to be more than 90% until fermentation is finished. A fermentation medium is composed of glucose, yeast extract, sodium chloride, K2HPO4, MgSO4, MnSO4 and water, and the initial pH value is 7.2-7.5; the supplementary liquid is composed of glucose and yeast extract, and the initial pH value is 7.2-7.5. The bacillus subtilis growth acceleration and endospore generation culture method is used for bacillus subtilis culture.

Description

A kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma
Technical field
The invention belongs to technical field of microbial fermentation; Be specifically related to a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma.
Background technology
Subtilis is a kind of aerobic bacteria, is also a kind of important industrial strain, has been widely used in the fields such as herding, medicine, control of plant disease, has been with a wide range of applications, demonstrates huge social benefit and ecological benefits.Subtilis probiotics overwhelming majority product all plays a role with viable bacteria, and effective viable count is the important indicator weighing probiotics quality, must contain the effect of a considerable amount of viable count competence exertion in product.But probiotics is along with the prolongation of shelf time, and number of viable constantly reduces, over a period to come, its living bacteria count reduces affects its result of use.Subtilis is because of the nourishing body of its uniqueness---gemma, and make it have the physiological property of the poor environments such as opposing heat, drying, radiation, acid, alkali, the generation of gemma can extend the preservation period of its probiotics thus.But the active bacteria formulation of current widespread use is due to the instability of sporulation, and still short, the active shortcoming such as unstable of ubiquity preservation period, brings a lot of difficulty to practical application.
In the suitability for industrialized production of present stage, owing to being convenient to each factor that control effect is cultivated, liquid cultivation is made to become the main cultivation means of industrial fermentation.But in liquid state is cultivated, the sprout processing condition of spore of producing bacillus subtilis are wayward, and spore forming rate is low, meanwhile, often there is the problems such as fermentation period is long, viable count is low in fermenting process.
Summary of the invention
In the liquid fermenting process of subtilis, sporulation associated process conditions is wayward, spore forming rate is low for the present invention's solution, and the problem that fermentation period is long, viable count is low, obtain the spore forming rate of more than 95% while realizing subtilis high-density culture; Provide a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma.
A kind ofly in the present invention promote bacillus subtilis bacteria growing and produce the cultural method of gemma to carry out in the steps below: a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma carries out in the steps below:
The activation of step one, bacterial classification: single bacterium colony bacterial classification access test tube seed culture medium on picking inclined-plane, under 30 DEG C of conditions, cultivate 16h with the concussion of 70r/min speed obtain seed liquor, described test tube seed culture medium formula is by mass percentage as follows: 0.8% glucose, 0.5% yeast extract paste, 0.3% sodium-chlor, 0.2%K 2hPO 4, 0.2%CaCO 3,surplus is water, and initial pH value is 7.2-7.5;
Step 2, shake-flask seed are cultivated: loaded by seed culture medium in shaking flask, then by the seed liquor that 2% inoculum size access step one obtains, under temperature 30 DEG C, speed are 180r/min condition, 16h cultivated by shaking table again, obtain seed liquor, described seed culture based formulas is identical with the test tube seed culture medium described in step one;
Step 3, fermentation: fermention medium is placed in fermentor tank, then by the seed liquor that 1% inoculum size access obtains through step 2, then temperature be 30 DEG C, pH value is 7, dissolved oxygen stirs fermentation under to control (air flow general control is at 10-15L/min), tank pressure 0.01 ~ 0.03MPa and stirring velocity more than 70% be 250 ~ 400r/min condition;
Step 4, when fermentation to the residual sugar amount of thalline be 0.1% (general need 20-24h consuming time), start to add feed supplement liquid and residual sugar amount is controlled (the flow acceleration 3mL/min of feed supplement liquid) condition bottom fermentation between 0.1 ~ 0.2, fermentation 4h is continued after stopping feed supplement after feed supplement 5 ~ 7h, then adjust ph is 8.5-9 and is controlled more than 90% by dissolved oxygen, until fermentation ends (the general time is 3-5h);
Wherein the formula by mass percentage of fermention medium described in step 3 is as follows: 0.5% ~ 1.0% glucose, 0.2% ~ 0.8% yeast extract paste, 0.1% ~ 0.5% sodium-chlor, 0.1% ~ 0.4%K 2hPO 4, 0.01% ~ 0.05%MgSO 4, 0.01% ~ 0.02%MnSO 4, surplus is water, initial pH value is 7.2-7.5;
Described in step 4, feed supplement liquid formula is by mass percentage as follows: glucose 8% ~ 12%, yeast extract paste 3% ~ 5%, and initial pH value is 7.2-7.5.
The present invention, by the strict control to fermentation of bacillus subtilis technique, supplements specific feed supplement liquid in good time, extends the logarithmic phase of bacterial strain, make its viable count reach 9,000,000,000/mL; By the formation jointly controlling stable promotion bacillus subtilis spore to pH and dissolved oxygen, rate of formation reaches more than 95%.
Improve spore forming rate while the present invention realizes the high-density culture of subtilis, extend its preparation preservation period and increase its stabilizing active, preservation period is more than 5 years.
Embodiment
Adopt the subtilis of deposit number CGMCC No.10603 to cultivate in present embodiment, concrete grammar is as follows:
The activation of step one, inclined-plane seed: single bacterium colony bacterial classification access test tube seed culture medium (5mL) on picking inclined-plane, 30 DEG C of concussions (70rpm) cultivate 16h.Test tube seed culture medium forms: glucose 8g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, K 2hPO 42g/L, CaCO 32g/L, initial pH are 7.2-7.5.
The cultivation of step 2, seed: shake-flask seed substratum liquid amount 50/500mL, by 2% inoculum size access test tube seed liquor, 30 DEG C of shaking table 180rpm cultivate 16h, obtain seed liquor; Shake-flask seed substratum is with test tube seed culture medium.
Step 3, batch fermentation: fermention medium (glucose 8g/L, yeast extract paste 5g/L, sodium-chlor 3g/L, K 2hPO 42g/L, MgSO 40.2g/L, MnSO 40.1g/L, initial pH are 7.2-7.5) liquid amount 10/20L, by 1% inoculum size access shake-flask seed liquid, 30 DEG C, pH value 7, air flow 10-15L/min, tank pressure 0.01-0.03MPa, stir 50-600rpm fermentation, dissolved oxygen controls more than 70%;
Step 4, ferment to logarithmic growth middle and later periods (residual sugar amount is about the 0.1%) 6h of thalline, start fed-batch fermentation, feed supplement liquid (glucose 10%, yeast extract paste 4%, initial pH 7.2-7.5) flow acceleration 3mL/min, residual sugar amount controls between 0.1-0.2, feed supplement 7h, stop feed supplement continuing fermentation 4h, then adjust ph is 8.5-9, improve dissolved oxygen to more than 90%, until fermentation ends (3-5h).
The viable count of present embodiment subtilis can reach 9,000,000,000/mL; Rate of formation reaches more than 95%.Preservation period more than 5 years, when 5 years, viable count can reach 8,000,000,000/mL.

Claims (5)

1. promote bacillus subtilis bacteria growing and produce the cultural method of gemma, it is characterized in that a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma carries out in the steps below:
The activation of step one, bacterial classification: cultivate in single bacterium colony bacterial classification access substratum on picking inclined-plane;
Step 2, shake-flask seed are cultivated;
Step 3, fermentation: fermention medium is placed in fermentor tank, then by the seed liquor that 1% inoculum size access obtains through step 2, then temperature be 30 DEG C, pH value is 7, dissolved oxygen controls more than 70%, tank pressure 0.01 ~ 0.03MPa and stirring velocity stir fermentation under being 250 ~ 600r/min condition;
Step 4, when fermentation to the residual sugar amount of thalline be 0.1%, start to add feed supplement liquid and residual sugar amount is controlled condition bottom fermentation between 0.1 ~ 0.2, continue fermentation 4h after stopping feed supplement after feed supplement 5 ~ 7h, then adjust ph is 8.5-9 and is controlled more than 90% by dissolved oxygen, until fermentation ends;
Wherein the formula by mass percentage of fermention medium described in step 3 is as follows: 0.5% ~ 1.0% glucose, 0.2% ~ 0.8% yeast extract paste, 0.1% ~ 0.5% sodium-chlor, 0.1% ~ 0.4%K 2hPO 4, 0.01% ~ 0.05%MgSO 4, 0.01% ~ 0.02%MnSO 4, surplus is water, initial pH value is 7.2-7.5;
Described in step 4, feed supplement liquid formula is by mass percentage as follows: glucose 8% ~ 12%, yeast extract paste 3% ~ 5%, and initial pH value is 7.2-7.5.
2. a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma according to claim 1, is characterized in that
In step one, the activation of bacterial classification is undertaken by following operation: single bacterium colony bacterial classification access test tube seed culture medium on picking inclined-plane, under 30 DEG C of conditions, cultivate 16h with the concussion of 70r/min speed obtain seed liquor, described test tube seed culture medium formula is by mass percentage as follows: 0.8% glucose, 0.5% yeast extract paste, 0.3% sodium-chlor, 0.2%K 2hPO 4, 0.2%CaCO 3,surplus is water, and initial pH value is 7.2-7.5.
3. a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma according to claim 1, it is characterized in that in step 2, shake-flask seed cultivation is undertaken by following operation: loaded by seed culture medium in shaking flask, then by the seed liquor that 2% inoculum size access step one obtains, under temperature 30 DEG C, speed are 180r/min condition, 16h cultivated by shaking table again, obtain seed liquor, described seed culture medium formula is by mass percentage as follows: 0.8% glucose, 0.5% yeast extract paste, 0.3% sodium-chlor, 0.2%K 2hPO 4, 0.2%CaCO 3, surplus is water, initial pH value is 7.2-7.5.
4. a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma according to claim 1, is characterized in that step 3 fermention medium formula is by mass percentage as follows: 0.8% glucose, 0.5% yeast extract paste, 0.3% sodium-chlor, 0.2%K 2hPO 4, 0.02%MgSO 4, 0.01%MnSO 4, surplus is water, initial pH value is 7.2-7.5.
5. a kind of cultural method promoting bacillus subtilis bacteria growing and produce gemma according to claim 1, it is characterized in that described in step 4, feed supplement liquid formula is by mass percentage as follows: glucose 10%, yeast extract paste 4%, initial pH value is 7.2-7.5.
CN201510345610.XA 2015-06-19 2015-06-19 Bacillus subtilis growth acceleration and endospore generation culture method Pending CN104894032A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105907661A (en) * 2016-04-08 2016-08-31 中国农业大学 Industrial fermentation method for Bacillus subtillis
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
CN106754477A (en) * 2016-11-23 2017-05-31 山东省农业科学院农业资源与环境研究所 The continuous cultural method and special fermentation system of a kind of bacillus licheniformis
CN106947715A (en) * 2017-03-23 2017-07-14 中国科学院青岛生物能源与过程研究所 A kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate

Citations (1)

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CN103667159A (en) * 2013-12-30 2014-03-26 广东海纳川药业股份有限公司 High-density culture method for bacilli and culture medium

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CN103667159A (en) * 2013-12-30 2014-03-26 广东海纳川药业股份有限公司 High-density culture method for bacilli and culture medium

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105907661A (en) * 2016-04-08 2016-08-31 中国农业大学 Industrial fermentation method for Bacillus subtillis
CN105907661B (en) * 2016-04-08 2020-01-21 中国农业大学 Industrial fermentation method of bacillus subtilis
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
CN106754477A (en) * 2016-11-23 2017-05-31 山东省农业科学院农业资源与环境研究所 The continuous cultural method and special fermentation system of a kind of bacillus licheniformis
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
CN106754477B (en) * 2016-11-23 2020-12-01 山东省农业科学院农业资源与环境研究所 Continuous culture method of bacillus licheniformis and special fermentation system
CN106947715A (en) * 2017-03-23 2017-07-14 中国科学院青岛生物能源与过程研究所 A kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate

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