CN106947715A - A kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate - Google Patents
A kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate Download PDFInfo
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Abstract
The invention provides a kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate, belong to technical field of microbial fermentation.Low for the current fermentation of bacillus subtilis viable bacteria level of solution, the problems such as sporulation is unstable, the present invention provides following scheme：Seed liquor is inoculated in the sterilising medium of fermentation tank, distribution ferment period control temperature fermentation：34 37 DEG C of cultures 4h, 35 40 DEG C of fermentation 8h, 37 40 DEG C to terminating；Distribute ferment period control air quantity：Ventilation 0.3 0.6vvm fermentation 8h, the control 1.0vvm of ventilation 0.4 are controlled to terminating；The duration that ferments is more than 12h；Ferment pH6.0 7.0.The method fermented and cultured 24h provided using the present invention, final bacillus subtilis viable count is up to 1.6 × 1010Cfu/ml, gemma rate reaches 96%.This fermentation process can be widely applied to bacillus subtilis viable bacteria fermentation field.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to a kind of fermentation of bacillus subtilis method.
Technical background
Bacillus subtilis (Bacillus subtilis) is the leather that a class is distributed widely in various different living environments
Lan Shi it is positive it is shaft-like support type bacterium well, can produce endogenous spore, heat-resisting strong stress resistance, be widely used in herding, medicine,
The fields such as control of plant disease, are with a wide range of applications, and show huge social benefit and ecological benefits.Withered grass gemma
Bacillus probiotics overwhelming majority product is all played a role with viable bacteria, and living bacteria count is to weigh probiotics quality
Important indicator, the effect of a considerable amount of viable count competence exertions must be contained in product.But probiotics is with preservation
Time lengthening, viable bacteria amount is reduced, over a period to come, its living bacteria count influence using effect.However, be widely used at present
Active bacteria formulation is low due to fermentation viable bacteria level, and sporulation is unstable to bring difficulty to practical application.At present, bacillus subtilis
Active bacteria formulation fermentation gained bacillus subtilis viable count highest only up to 1.0 × 1010Cfu/ml, gemma rate 90%, due to existing
Not enough above-mentioned viable count and the gemma rate of microbial fermentation technology is difficult to improve again.
Therefore, the higher product of living bacteria count how is obtained, is technique most crucial during feeding micro-ecological preparation is produced
Problem.This is also the hot issue in fermentation industry field.
The content of the invention
The purpose of the present invention is exactly the i.e. active bacteria formulation fermentation viable bacteria level in order to overcome above-mentioned existing technological deficiency
Low, there is provided a kind of high viable count, the fermentation side of the bacillus subtilis of high spore forming rate for the problems such as sporulation is unstable
Method.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate, including：Add and go out in fermentation tank
The liquid fermentation medium of bacterium, seed liquor is inoculated in the sterilising medium of fermentation tank according to mass percent 5%, cultivates bar
Part is：Started at from fermentation initial time, distribution ferment period control temperature fermentation：Control 34-37 DEG C of culture 4h of temperature, control temperature
35-40 DEG C of fermentation 8h of degree, the 37-40 DEG C of fermentation of control temperature is until fermentation ends；Started at from fermentation initial time, point fermentation time
Section control air quantity：Ventilation 0.3vvm-0.6vvm fermentation 8h are controlled, control ventilation 0.4vvm-1.0vvm fermentations are until fermentation
Terminate；Total duration of fermenting is more than 12h；Fermentation process pH6.0-7.0；Speed of agitator 300rpm-800rpm.(adjusted according to oxygen dissolving value
Whole, oxygen dissolving value rise reduces speed of agitator；Oxygen dissolving value is reduced, and raises speed of agitator).
It is preferred that, the liquid fermentation medium constituent includes according to mass percent：Carbon source 3%-5%, nitrogen source
3%-6%, potassium dihydrogen phosphate 0.01%-0.03%, bitter salt 0.01%-0.02%, alpha-amylase 0.02%, its
Yu Weishui, liquid fermentation medium is with postponing, need sterilizing before use.
It is preferred that, the carbon source is the one or more of corn flour, sucrose, lactose or soluble starch；Told nitrogen source is
Bean cake powder, yeast extract, beef extract, the one or more of peptone or corn steep liquor.
It is preferred that, the liquid fermentation medium volume accounts for the 40% of fermenter volume.
It is preferred that, the preparation method of the seed liquor is：Strain is directly inoculated in eggplant bottle from sand pipe picking colony
Inclined-plane, 37 DEG C of cultivation temperature, incubation time 72h；With inoculation shovel by whole eggplant bottle inclined plane inoculating in equipped with bead and sterile
In the triangular flask of water, shaking table 37 DEG C of temperature of control is placed, 2h, rotating speed 180rpm is cultivated.
It is preferred that, described bottle inclined plane culture medium of eggplant constituent includes according to mass percent：Yeast extract
0.5%, tryptone 1%, sodium chloride 1%, agar 1.5%, natural pH, remaining is water, need to be in 121- with postponing, before use
Sterilize 40min at 123 DEG C.
Beneficial effect：
1st, using new culture medium prescription, bacillus subtilis viable bacteria fermentation cycle time, viable count are significantly improved, sent out
Ferment 24h is up to 1.6 × 1010Cfu/ml, nearly 60% is improved than prior art highest level, achieves unexpected skill
Art effect.
2nd, in fermentation process, the technical scheme of ventilation is controlled using time segment, makes fermentation of bacillus subtilis viable bacteria
Number is significantly improved, and fermentation 24h is up to 1.6 × 1010Cfu/ml, nearly 60%, acquirement are improved than prior art highest level
Unexpected technique effect.
3rd, in fermentation process, the technical scheme of temperature, gained fermentation of bacillus subtilis 24h buds are controlled using time segment
Spore rate reaches as high as 96%, significantly improves the gemma number and product quality of product.
Specific embodiment：
In order to be best understood from the present invention, with embodiment, the invention will be further described below.
Embodiment 1：
1) prepared by bacterial strain：By the above-mentioned preferable mutant strain of secondary screening effect, in inclined-plane LB culture mediums, (slant medium is according to matter
Percentage is measured, is made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C,
0.11Mpa sterilize 20min) culture 3 days after, carry out sand tube preservation.37 DEG C of cultivation temperature, incubation time 72h.
2) seed culture：Sand pipe is directly inoculated in into eggplant bottle inclined-plane, and (bottle inclined plane culture medium of eggplant is according to quality percentage
Than being made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C, 0.11Mpa
Sterilize 20min), 37 DEG C of cultivation temperature, incubation time 3 days prepares seed liquor.
3) fermentation tank is supported：Using 5L fermentation cylinder for fermentation culture volumes 2L (fermentation medium according to mass percent, by
Following composition composition：Carbon source corn flour 3%, nitrogen source dregs of beans 3%, potassium dihydrogen phosphate 0.01%, bitter salt 0.01% is high
Warm amylase 0.02%, adjusts pH6.0,121-123 DEG C, sterilize 40min), the seed liquor prepared is inoculated in 5L fermentation tanks
Sterilising medium in.
Condition of culture is that distribution ferment period control temperature, fermentation starts, 34 DEG C of culture 4h of control temperature；35 DEG C of temperature
Ferment 8h；The fermentation of 37 DEG C of temperature of control is to terminating.Distribute ferment period control air quantity：Fermentation starts, control ventilation 0.3vvm
Cultivate 8h；Ventilation 0.4vvm terminates to culture.Fermentation processes pH6.0, control speed of agitator 300rpm.The duration that ferments is total to
24h, terminates collection fermentation culture after fermentation and obtains high gemma rate bacillus subtilis fluid product, mixed ware valve detection is lived
Bacterium number 1.4 × 1010Cfu/ml, gemma rate 94%.The present embodiment uses new culture medium prescription, sends out bacillus subtilis viable bacteria
Ferment cycle time, viable count are significantly improved, and are controlled the technical scheme of ventilation in fermentation process using time segment, are made withered grass bud
Spore bacillus fermentation viable count is significantly improved, and fermentation 24h reaches viable count 1.4 × 1010cfu/ml.Using between timesharing in fermentation process
The technical scheme of section control temperature, gained fermentation of bacillus subtilis 24h gemma rate significantly improves the gemma number of product up to 96%
And product quality.
Embodiment 2
1) prepared by bacterial strain：By the above-mentioned preferable mutant strain of secondary screening effect, in inclined-plane LB culture mediums, (slant medium is according to matter
Percentage is measured, is made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C,
0.11Mpa sterilize 20min) culture 3 days after, carry out sand tube preservation.37 DEG C of cultivation temperature, incubation time 72h.
2) seed culture：Sand pipe is directly inoculated in into eggplant bottle inclined-plane, and (bottle inclined plane culture medium of eggplant is according to quality percentage
Than being made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C, 0.11Mpa
Sterilize 20min), 37 DEG C of cultivation temperature, incubation time 3 days.
3) fermentation tank is supported：Using 5L fermentation cylinder for fermentation culture volumes 2L (fermentation medium according to mass percent, by
Following composition composition：Carbon source corn flour 5%, nitrogen source dregs of beans 6%, potassium dihydrogen phosphate 0.03%, bitter salt 0.02% is high
Warm amylase 0.02%, adjusts pH6.5,121-123 DEG C, sterilize 40min), the seed liquor prepared is inoculated in 5L fermentation tanks
Sterilising medium in.
Condition of culture is that distribution ferment period control temperature, fermentation starts, 37 DEG C of culture 4h of control temperature；40 DEG C of temperature
Ferment 8h；The fermentation of 40 DEG C of temperature of control is to terminating.Distribute ferment period control air quantity：Fermentation starts, control ventilation 0.6vvm
Cultivate 8h；Ventilation 1.0vvm terminates to culture.Fermentation processes pH7.0, control speed of agitator 800rpm.The duration that ferments is total to
24h, terminates collection fermentation culture after fermentation and obtains high gemma rate bacillus subtilis fluid product, mixed ware valve detection is lived
Bacterium number 1.5 × 1010Cfu/ml, gemma rate 96%.The present embodiment uses new culture medium prescription, sends out bacillus subtilis viable bacteria
Ferment cycle time, viable count are significantly improved, and are controlled the technical scheme of ventilation in fermentation process using time segment, are made withered grass bud
Spore bacillus fermentation viable count is significantly improved, and fermentation 24h reaches viable count 1.6 × 1010cfu/ml.Using between timesharing in fermentation process
The technical scheme of section control temperature, gained fermentation of bacillus subtilis 24h gemma rate significantly improves the gemma number of product up to 96%
And product quality.
Embodiment 3
Difference with embodiment 2 is：Carbon source corn flour is replaced by sucrose, and mass percent is constant, terminates to receive after fermentation
Collection fermentation culture obtains high gemma rate bacillus subtilis fluid product, and mixed ware valve detection obtains viable count 1.2 × 1010cfu/
Ml, gemma rate 95%.The present embodiment uses new culture medium prescription, makes bacillus subtilis viable bacteria fermentation cycle time, viable bacteria
Number is significantly improved, and is controlled the technical scheme of ventilation in fermentation process using time segment, is made fermentation of bacillus subtilis viable bacteria
Number is significantly improved, and fermentation 24h reaches viable count 1.2 × 1010cfu/ml.The skill of temperature is controlled in fermentation process using time segment
Art scheme, gained fermentation of bacillus subtilis 24h gemma rate significantly improves the gemma number and product quality of product up to 95%.
Embodiment 4
Difference with embodiment 2 is：Carbon source corn flour is replaced by lactose, and mass percent is constant, terminates to receive after fermentation
Collection fermentation culture obtains high gemma rate bacillus subtilis fluid product, and mixed ware valve detection obtains viable count 1.5 × 1010cfu/
Ml, gemma rate 94%.The present embodiment uses new culture medium prescription, makes bacillus subtilis viable bacteria fermentation cycle time, viable bacteria
Number is significantly improved, and is controlled the technical scheme of ventilation in fermentation process using time segment, is made fermentation of bacillus subtilis viable bacteria
Number is significantly improved, and fermentation 24h reaches viable count 1.1 × 1010cfu/ml.The skill of temperature is controlled in fermentation process using time segment
Art scheme, gained fermentation of bacillus subtilis 24h gemma rate significantly improves the gemma number and product quality of product up to 94%.
Embodiment 5
Difference with embodiment 2 is：Carbon source corn flour is replaced by soluble starch, and mass percent is constant, terminates hair
Fermentation culture is collected after ferment and obtains high gemma rate bacillus subtilis fluid product, mixed ware valve detection obtain viable count 1.3 ×
1010Cfu/ml, gemma rate 95%.The present embodiment uses new culture medium prescription, the bacillus subtilis viable bacteria fermentation cycle is contracted
Short, viable count is significantly improved, and is controlled the technical scheme of ventilation in fermentation process using time segment, is sent out bacillus subtilis
Ferment viable count is significantly improved, and fermentation 24h reaches viable count 1.3 × 1010cfu/ml.Temperature is controlled using time segment in fermentation process
The technical scheme of degree, gained fermentation of bacillus subtilis 24h gemma rate significantly improves the gemma number and product matter of product up to 95%
Amount.
Embodiment 6：
1) prepared by bacterial strain：By the above-mentioned preferable mutant strain of secondary screening effect, in inclined-plane LB culture mediums, (slant medium is according to matter
Percentage is measured, is made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C,
0.11Mpa sterilize 20min) culture 3 days after, carry out sand tube preservation.37 DEG C of cultivation temperature, incubation time 72h.
2) seed culture：Sand pipe is directly inoculated in into eggplant bottle inclined-plane, and (bottle inclined plane culture medium of eggplant is according to quality percentage
Than being made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C, 0.11Mpa
Sterilize 20min), 37 DEG C of cultivation temperature, incubation time 3 days.
3) fermentation tank is supported：Using 5L fermentation cylinder for fermentation culture volumes 2L (fermentation medium according to mass percent, by
Following composition composition：Carbon source corn flour 4%, nitrogen source beef extract 6%, potassium dihydrogen phosphate 0.01%, bitter salt 0.02%,
Alpha-amylase 0.02%, adjusts pH6.5,121-123 DEG C, sterilize 40min), by the seed liquor prepared (cultured eggplant
Inclined-plane is wiped off with transfer needle pin, is positioned in the triangular flask equipped with bead and sterilized water, places shaking table culture 2h, rotating speed
In the sterilising medium for 180rpm) being inoculated in 5L fermentation tanks.Condition of culture is that distribution ferment period control temperature, fermentation starts,
Control 34-37 DEG C of culture 4h of temperature；35-40 DEG C of fermentation 8h of temperature；The fermentation of 37-40 DEG C of temperature of control is to terminating.Divide fermentation time
Section control air quantity：Fermentation starts, control ventilation 0.6vvm cultures 8h；Ventilation 1.0vvm terminates to culture.Fermentation process control
PH6.5 processed, control speed of agitator 800rpm.Ferment the common 24h of duration, terminates collection fermentation culture after fermentation and obtains high gemma rate
Bacillus subtilis fluid product, mixed ware valve detection obtains viable count 1.5 × 1010Cfu/ml, gemma rate 95%.The present embodiment is adopted
With new culture medium prescription, bacillus subtilis viable bacteria fermentation cycle time, viable count are significantly improved, is used in fermentation process
Time segment controls the technical scheme of ventilation, significantly improves fermentation of bacillus subtilis viable count, and fermentation 24h reaches viable bacteria
Number 1.5 × 1010cfu/ml.The technical scheme of temperature, gained bacillus subtilis hair are controlled in fermentation process using time segment
Ferment 24h gemma rate significantly improves the gemma number and product quality of product up to 95%.
Embodiment 7：
1) prepared by bacterial strain：By the above-mentioned preferable mutant strain of secondary screening effect, in inclined-plane LB culture mediums, (slant medium is according to matter
Percentage is measured, is made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C,
0.11Mpa sterilize 20min) culture 3 days after, carry out sand tube preservation.37 DEG C of cultivation temperature, incubation time 72h.
2) seed culture：Sand pipe is directly inoculated in into eggplant bottle inclined-plane, and (bottle inclined plane culture medium of eggplant is according to quality percentage
Than being made up of following composition：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, natural pH, 121 DEG C, 0.11Mpa
Sterilize 20min), 37 DEG C of cultivation temperature, incubation time 3 days.
3) fermentation tank is supported：Using 5L fermentation cylinder for fermentation culture volumes 2L (fermentation medium according to mass percent, by
Following composition composition：Corn flour 4%, corn steep liquor 6%, potassium dihydrogen phosphate 0.01%, bitter salt 0.02%, high-temperature starch
Enzyme 0.02%, adjusts pH6.5,121-123 DEG C, sterilize 40min), by the seed liquor prepared, (cultured eggplant inclined-plane is with connecing
Plant pin pin to wipe off, be positioned in the triangular flask equipped with bead and sterilized water, place shaking table culture 2h, rotating speed 180rpm) inoculation
In the sterilising medium of 5L fermentation tanks.Condition of culture is that distribution ferment period control temperature, fermentation starts, control temperature 34-
37 DEG C of culture 4h；35-40 DEG C of fermentation 8h of temperature；The fermentation of 37-40 DEG C of temperature of control is to terminating.Distribute ferment period control air quantity：
Fermentation starts, control ventilation 0.6vvm cultures 8h；Ventilation 1.0vvm terminates to culture.Fermentation processes pH6.0-7.0,
Control speed of agitator 800rpm.Ferment the common 24h of duration, terminates collection fermentation culture after fermentation and obtains high gemma rate withered grass gemma
Bacillus fluid product, mixed ware valve detection obtains viable count 1.4 × 1010Cfu/ml, gemma rate 94%.The present embodiment uses new training
Based formulas is supported, bacillus subtilis viable bacteria fermentation cycle time, viable count is significantly improved, time segment is used in fermentation process
Control ventilation technical scheme, significantly improve fermentation of bacillus subtilis viable count, fermentation 24h reach viable count 1.4 ×
1010cfu/ml.The technical scheme of temperature, gained fermentation of bacillus subtilis 24h buds are controlled in fermentation process using time segment
Spore rate significantly improves the gemma number and product quality of product up to 94%.
The above-mentioned description to embodiment is understood that for the ease of those skilled in the art and using hair
It is bright.Person skilled in the art obviously can readily make various modifications to these case study on implementation, and illustrating herein
General Principle be applied to other and apply in example and without performing creative labour.Therefore, the invention is not restricted to above-described embodiment, this
Art personnel are according to the announcement of the present invention, and the improvement and modification not departed from made by scope all should be in the present invention
Protection domain in.
Claims (6)
1. a kind of high viable count, the fermentation process of the bacillus subtilis of high spore forming rate, it is characterised in that：Add in fermentation tank
Enter the liquid fermentation medium of sterilizing, seed liquor is inoculated in the sterilising medium of fermentation tank, condition of culture is：From fermentation
Time beginning starts at, distribution ferment period control temperature fermentation：Control 34-37 DEG C of culture 4h of temperature, the 35-40 DEG C of fermentation of control temperature
8h, the 37-40 DEG C of fermentation of control temperature is until fermentation ends；Started at from fermentation initial time, distribution ferment period control air quantity：Control
Ventilation 0.3vvm-0.6vvm fermentations 8h processed, the 0.4vvm-1.0vvm fermentations of control ventilation are until fermentation ends；When fermenting total
It is long to be more than 12h；Fermentation process pH6.0-7.0；Speed of agitator 300rpm-800rpm.
2. fermentation process according to claim 1, it is characterised in that：The liquid fermentation medium constituent is according to matter
Amount percentage includes：Carbon source 3%-5%, nitrogen source 3%-6%, potassium dihydrogen phosphate 0.01%-0.03%, bitter salt
0.01%-0.02%, alpha-amylase 0.02%.
3. fermentation process according to claim 2, it is characterised in that：The carbon source is corn flour, sucrose, lactose or solvable
The one or more of property starch；The nitrogen source is the one or more of bean cake powder, yeast extract, beef extract, peptone or corn steep liquor.
4. fermentation process according to claim 1, it is characterised in that：Seed liquor is inoculated in hair according to mass percent 5%
In the sterilising medium of fermentation tank；The liquid fermentation medium volume accounts for the 40% of fermenter volume.
5. fermentation process according to claim 1, it is characterised in that：The preparation method of the seed liquor is：By strain from
Sand pipe is directly inoculated in eggplant bottle inclined-plane, 37 DEG C of cultivation temperature, incubation time 72h；With transfer needle from eggplant bottle inclined-plane by bacterium
Plant and be inoculated in the triangular flask equipped with bead and sterilized water, place shaking table culture, 37 DEG C of temperature, incubation time 2h, rotating speed
180rpm。
6. fermentation process according to claim 5, it is characterised in that：Described bottle inclined plane culture medium of eggplant constituent is pressed
Include according to mass percent：Yeast extract 0.5%, tryptone 1%, sodium chloride 1%, agar 1.5%, natural pH.
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| CN112175854A (en) * | 2019-07-05 | 2021-01-05 | 中粮生物化学（安徽）股份有限公司 | High-density fermentation method for rapidly producing spores by using bacillus subtilis |
| CN112175854B (en) * | 2019-07-05 | 2022-11-22 | 中粮生物科技股份有限公司 | High-density fermentation method for rapidly producing spores by using bacillus subtilis |
| CN110305812B (en) * | 2019-07-05 | 2023-06-13 | 山东苏柯汉生物工程股份有限公司 | Bacillus licheniformis fermentation culture process |
| CN111073837A (en) * | 2019-12-31 | 2020-04-28 | 盐城工学院 | Fermentation method for promoting Paenibacillus polymyxa to produce spores |
| CN111073837B (en) * | 2019-12-31 | 2023-08-25 | 盐城工学院 | Fermentation method for promoting Paenibacillus polymyxa to produce spores |
| CN114276952A (en) * | 2021-12-06 | 2022-04-05 | 湖北工业大学 | Method for improving spore formation efficiency of bacillus licheniformis |
| CN114276952B (en) * | 2021-12-06 | 2023-08-18 | 湖北工业大学 | Method for improving bacillus licheniformis spore formation efficiency |
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Application publication date: 20170714 |