CN107988134A - A kind of strain domestication method for improving bacillus production gemma rate - Google Patents
A kind of strain domestication method for improving bacillus production gemma rate Download PDFInfo
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- CN107988134A CN107988134A CN201810052769.6A CN201810052769A CN107988134A CN 107988134 A CN107988134 A CN 107988134A CN 201810052769 A CN201810052769 A CN 201810052769A CN 107988134 A CN107988134 A CN 107988134A
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Abstract
The invention discloses a kind of strain domestication method for improving bacillus production gemma rate, comprise the following steps：Bacillus is activated；The order progress secondary culture that strain after activation is cultivated according to nutrient concentrations from normal incubation medium to barren culture medium, are then further added by into normal incubation medium successively, culture medium set more than 3 according to concentration, and continuous passage 3 is more than generation.The strain domestication method provided by the invention for improving bacillus production gemma rate, largely shortens the culture domestication time, easy to operate, the method according to the invention, can make the production gemma rate of bacillus bring up to 96%.
Description
Technical field
The present invention relates to microbial technology field, relates particularly to a kind of strain for improving bacillus production gemma rate and tames and dociles
Change method.
Background technology
With probiotics become feed addictive research hot spot, advantage of the probiotics preparation in animal feeding by
Step shows.But most of probiotics are poor there are resistance, heat labile shortcoming, preservation, transport to preparation all bring compared with
It is big difficult.Bacillus is a kind of aerobic production bacillus being widely present in nature, and gemma is bacteriotrophy body must
When needing nutriment will exhaust, the water content that is formed in the cell is extremely low, resistance is extremely strong hypopus.
Research shows that gemma caused by bacillus has heat-resisting, drought-enduring, uvioresistant and organic solvent etc. are a variety of to resist
Inverse property, due to the resistance that gemma is extremely strong, during interchangeable manufacturing produces high temperature drying, it is ensured that microbial bacterial agent
Activity, improve its product quality, and it is to person poultry harmless, it is free from environmental pollution, so being obtained in animal husbandry, feedstuff industry
Extensive use.At present, screening obtains probiotic bacillus kind generally existing bud in industrial processes from natural habitat
The problems such as spore formation rate is not high, application effect is unstable.How to improve producing bacillus subtilis gemma rate and have become limitation bud
One of bottleneck problem of spore bacillus probiotics development.
Chinese patent literature CN102181389A discloses a kind of method of breeding Bacillaceae with high spore yield, this method
It is the thermal stability characteristics according to gemma, the means cultivated after being handled using stepped heating, select the gemma of high yield gemma rate
Bacillus.Its step includes：Bacillus activation, level-one heating, level-one culture, two level heating, two level culture, three-level
Heating, third stage culture and collection, obtain the high yield gemma rate bacillus.Although the above method improves bacillus
Production gemma rate, but the method needs longer incubation time, by activating and pass through third stage culture, at least 3-5 days every time, trains
The foster time for once at least needing 2 weeks, the time of culture domestication is elongated, and after culture, it is also necessary to carry out different journeys
The heat treatment of degree is, it is necessary to which careful set temperature and time, add control cost.So establish it is a kind of quick, efficiently and
The strain domestication method that technique simply improves bacillus production gemma rate is very necessary.
The content of the invention
The technical problem to be solved in the present invention is in order to overcome the gemma of breeding high-yield gemma rate existing in the prior art
A kind of the problems such as bacillus incubation time is long, complicated, there is provided strain domestication method for improving bacillus production gemma rate.
To achieve the above object, the present invention provides following technical solution：
A kind of strain domestication method for improving bacillus production gemma rate, comprises the following steps：
Bacillus is activated；
By the strain after activation successively according to from normal incubation medium to barren culture medium, nutrient concentrations are then further added by
The order cultivated into normal incubation medium carries out secondary culture, and culture medium sets more than 3 according to concentration, 3 generation of continuous passage with
On.
In above-mentioned strain domestication method, the normal incubation medium is the culture medium of 80-100% original concentrations, described barren
Culture medium is 12.5%-80% original concentration culture mediums.
In above-mentioned strain domestication method, it is described from normal incubation medium to barren culture medium again to normal incubation medium, progressively set
3-9 concentration gradient is put, from normal incubation medium to barren culture medium, latter concentration is the 12.5%-80% of previous concentration；From poor
For lean culture medium to normal incubation medium, previous concentration is the 12.5%-80% of latter concentration.
In above-mentioned strain domestication method, it is described from normal incubation medium to barren culture medium again to normal incubation medium, progressively set
3-7 concentration gradient is put, from normal incubation medium to barren culture medium, latter concentration is the 12.5%-80% of previous concentration；From poor
For lean culture medium to normal incubation medium, previous concentration is the 12.5%-80% of latter concentration.
In above-mentioned strain domestication method, it is described from normal incubation medium to barren culture medium again to the order of normal incubation medium
For：Original concentration culture medium, 50% original concentration culture medium, 25% original concentration culture medium, 12.5% original concentration culture medium,
25% original concentration culture medium, 50% original concentration culture medium, original concentration culture medium.
In above-mentioned strain domestication method, the original concentration culture medium is：Per 1000mL, following component is included：Tryptose
Peptone 8-12g, yeast extract 1.5-3g, glucose 1.5-4g, beef extract 2-5g, sodium chloride 3-6g, disodium hydrogen phosphate 2.5-4g, seven
Water magnesium sulfate 0.5-1.5g, pH value 7.0-7.4.
In above-mentioned strain domestication method, it is preferred that the original concentration culture medium is：Per 1000mL, following component is included：
Tryptone 10g, yeast extract 2g, glucose 2g, beef extract 3g, sodium chloride 4g, disodium hydrogen phosphate 3g, epsom salt
1.0g, pH value 7.2.
In above-mentioned strain domestication method, after every generation terminates culture, heating water bath, is then passed on next time again.
In above-mentioned strain domestication method, the method for the heating water bath is：80-100 DEG C, heat 5-20min.
In above-mentioned strain domestication method, the condition of the secondary culture is：Shake flask fermentation, fermentation temperature are 30-37 DEG C, hair
The ferment time is 30-48h, rotating speed 180-220r/min, pH value 7.0-7.4, it is preferred that fermentation temperature is 37 DEG C, during fermentation
Between be 36h, rotating speed 200r/min, pH value 7.0.
In above-mentioned strain domestication method, the bacillus is that can form the bacillus or coccus of gemma, including bacillus
Category, Sporolactobacillus, fusobacterium, Desulfotomaculum and Sporosarcina, it is preferred that for bacillus subtilis,
Clothing bacillus, bacillus megaterium, bacillus coagulans, bacillus cereus, bafillus natto or clostridium butyricum.
In above-mentioned strain domestication method, the method for the secondary culture is：It is inoculated with every time with the inoculum concentration of volume ratio 2-5%
Into culture medium.
In above-mentioned strain domestication method, the method for the bacillus activation is：By bacillus in 37 DEG C of Shaking cultures
Cultivate 10-15h, rotating speed 200r/min.
In above-mentioned strain domestication method, often it is commissioned to train after supporting, using the method for plate culture count detection bacterium sum；Water-bath
After heating, passage of future generation is carried out using bacterium solution as seed liquor, using the growing amount of the method for plate culture count detection gemma.
Bacterium sum and gemma detection with culture medium are nutrient agar (NA) culture medium, and specific component is (g/L)：Peptone
10g, beef extract 3g, sodium chloride 5g, agar 15g.
X% original concentration culture mediums described herein, refer to nutriment be original concentration culture medium X% or
More than X%, for example, 50%, 25%, 12.5% concentration cultures concentration be respectively above-mentioned original culture medium concentration 50%,
25%th, 12.5%, i.e., in the culture medium of same volume, the dosage of each nutriment be respectively original culture medium 50%,
25%th, 12.5%.
The invention has the advantages that：
The strain domestication method provided by the invention for improving bacillus production gemma rate, by the way that strain is normally being cultivated
Base, reduces nutrient concentrations to barren culture medium, is then stepped up nutrient concentrations again to the order of normal incubation medium
Carry out secondary culture mode, using the nutriment for changing culture medium number, change strain existence environment, to improve bud
The production gemma rate of spore bacillus.The method of the present invention largely shortens the culture domestication time, easy to operate, according to the present invention
Method, the production gemma rate of bacillus can be made to bring up to 96%.
Embodiment
Below will the present invention is described in further detail by specific embodiment.It should be appreciated that this place
The embodiment of description is merely to illustrate and explain the present invention, and is not intended to limit the invention.Made in following embodiments
Reagent and instrument, are commercially available unless otherwise specified.
The Bacillus subtilis ANSB 060 used in the examples below, its preserving number is CGMCC No.3440, in CN
Disclosed in 101705203A；Bacillus subtilis ANSB01G, its preserving number is CGMCC No.4297, in CN 102181376A
Disclosed in；Bacillus subtilis ANSB471, its preserving number is CGMCC No.7344, disclosed in CN 103243047A.
Embodiment 1
1st, the preparation of seed liquor
The Bacillus subtilis ANSB 060 shake flask fermentation of preservation is done into seed liquor, in 37 DEG C of insulating box shake flask fermentation cultures
12h, rotating speed 200r/min, obtaining Bacillus subtilis ANSB 060 seed liquor, (viable bacteria concentration is about 109CFU/mL)
The formula of wherein fermentation medium is：Contain per 1000mL culture mediums：Tryptone 8g, yeast extract 3g, grape
Sugared 2g, beef extract 5g, sodium chloride 3g, disodium hydrogen phosphate 4g, epsom salt 1.5g, pH value 7.0.
After mentioned component is weighed well by formula, suitable water is dissolved in, then adds water constant volume, adjusts pH value, and pass through 121
DEG C steam sterilizing 20min, it is spare.
2nd, the strain passage culture of various concentrations culture medium
Above-mentioned Bacillus subtilis ANSB 060 seed liquor is taken with the inoculum concentration of 2% (volume ratio) to be inoculated into 50ml original dense
Spend in the 250ml triangular flasks of zymotic fluid (i.e. above-mentioned fermentation medium), fermentation temperature is 37 DEG C, pH value 7.0, rotating speed 200r/
Min, fermentation time 36h, obtains zymotic fluid A.
After taking the zymotic fluid A, 80 DEG C of heating water bath 15min of above-mentioned Bacillus subtilis ANSB 060, with 2% (volume ratio)
Inoculum concentration be inoculated into the 250ml triangular flasks of 50% original concentration zymotic fluids of 50ml, fermentation temperature be 37 DEG C, pH value 7.0,
Rotating speed 200r/min, fermentation time 36h, obtains zymotic fluid B.
Above-mentioned Bacillus subtilis ANSB 060 zymotic fluid B is taken, after 80 DEG C of heating water bath 15min, with 2% (volume ratio)
Inoculum concentration is inoculated into the 250ml triangular flasks of 50ml original concentration zymotic fluids, and fermentation temperature is 37 DEG C, pH value 7.0, rotating speed
200r/min, fermentation time 36h.
Zymotic fluid after each concentration cultures culture is used as next concentration culture after 80 DEG C of heating water bath 15min
The seed liquor of base.Secondary culture is carried out on original culture medium, 50% concentration cultures, original culture medium successively, continuously passes 3
Generation.
3rd, the calculating of gemma rate
Often it is commissioned to train after supporting, using the method for plate culture count detection bacterium sum；By 80 DEG C of heating water bath 15min
Afterwards, using the growing amount of the method for plate culture count detection gemma.The calculation of wherein gemma rate is as follows：
Gemma rate (%)=(gemma number/total viable count) * 100%.
Total viable count and gemma detection are nutrient agar (NA) culture medium with culture medium, and specific component is (g/L)：Peptone
10g, beef extract 3g, sodium chloride 5g, agar 15g.
After 3 are commissioned to train and support, the production gemma rate of Bacillus subtilis ANSB 060 has brought up to 93% from 75%.
Embodiment 2
1st, the preparation of seed liquor
By the bacillus subtilis ANSB01G of preservation, shake flask fermentation does seed liquor, in 37 DEG C of insulating box shake culture 12h,
Rotating speed 200r/min, obtains bacillus subtilis ANSB01G seed liquors.(viable bacteria concentration is about 109CFU/mL)
The formula of wherein fermentation medium is：Contain per 1000mL culture mediums：Per tryptone 10g, yeast extract 2g, Portugal
Grape sugar 2g, beef extract 3g, sodium chloride 4g, disodium hydrogen phosphate 3g, epsom salt 1.0g, pH value 7.2.Preparation method is as implemented
1,121 DEG C of steam sterilizing 20min of example.
2nd, the strain passage culture of various concentrations culture medium
Above-mentioned bacillus subtilis ANSB01G seed liquors are taken with the inoculum concentration of 2% (volume ratio) to be inoculated into 50ml original dense
Spend in the 250ml triangular flasks of zymotic fluid (fermentation medium of above-mentioned " 1, the preparation of seed liquor in "), fermentation temperature is 37 DEG C, pH
Value 7.0, rotating speed 200r/min, fermentation time 36h, obtains zymotic fluid A.
Above-mentioned bacillus subtilis ANSB01G zymotic fluids A is taken, after 80 DEG C of heating water bath 15min, with 2% (volume ratio)
Inoculum concentration is inoculated into the 250ml triangular flasks of 50% original concentration zymotic fluids of 50ml, and fermentation temperature is 37 DEG C, and pH value 7.0, turns
Fast 200r/min, fermentation time 36h, obtains zymotic fluid B.The 50% concentration cultures concentration is the 1/ of original culture medium concentration
2,121 DEG C of steam sterilizing 20min.
Above-mentioned bacillus subtilis ANSB01G zymotic fluids B is taken, after 80 DEG C of heating water bath 15min, with 2% (volume ratio)
Inoculum concentration is inoculated into the 250ml triangular flasks of 25% original concentration zymotic fluids of 50ml, and fermentation temperature is 37 DEG C, and pH value 7.0, turns
Fast 200r/min, fermentation time 36h, obtains zymotic fluid C.The 25% concentration cultures concentration is the 1/ of original culture medium concentration
4,121 DEG C of steam sterilizing 20min.
Above-mentioned bacillus subtilis ANSB01G zymotic fluids C is taken, after 80 DEG C of heating water bath 15min, with 2% (volume ratio)
Inoculum concentration is inoculated into the 250ml triangular flasks of 12.5% original concentration zymotic fluids of 50ml, and fermentation temperature is 37 DEG C, pH value 7.0,
Rotating speed 200r/min, fermentation time 36h, obtains zymotic fluid D.The 12.5% concentration cultures concentration is original culture medium concentration
1/8,121 DEG C of steam sterilizing 20min.
According to above-mentioned fermented and cultured mode, successively on 25% concentration cultures, 50% concentration cultures, original culture medium
Secondary culture.Zymotic fluid after each concentration cultures culture is trained after 80 DEG C of heating water bath 15min as next concentration
Support the seed liquor of base.I.e. successively in original culture medium, 50% concentration cultures, 25% concentration cultures, 12.5% concentration culture
Base, 25% concentration cultures, 50% concentration cultures, 7 generation of secondary culture on original culture medium.
3rd, the calculating of gemma rate
Often it is commissioned to train after supporting, using the method for plate culture count detection bacterium sum；By 80 DEG C of heating water bath 15min
Afterwards, using the growing amount of the method for plate culture count detection gemma.The calculation of wherein gemma rate is as follows：
Gemma rate (%)=(gemma number/total viable count) * 100%.
Total viable count and gemma detection are nutrient agar (NA) culture medium with culture medium, and specific component is (g/L)：Peptone
10g, beef extract 3g, sodium chloride 5g, agar 15g.
After 7 are commissioned to train and support, the production gemma rate of bacillus subtilis ANSB01G has brought up to 96% from 71%.
Embodiment 3
1st, the preparation of seed liquor
By the bacillus subtilis ANSB471 of preservation, shake flask fermentation does seed liquor, in 37 DEG C of insulating box shake culture 12h,
Rotating speed 200r/min, obtaining bacillus subtilis ANSB471 seed liquors, (viable bacteria concentration is about 109CFU/mL)。
The formula of wherein fermentation medium is：Contain per 1000mL culture mediums：Tryptone 12g, yeast extract 1.5g, Portugal
Grape sugar 3g, beef extract 2g, sodium chloride 4g, disodium hydrogen phosphate 2.5g, epsom salt 0.5g, distilled water 1000ml, pH value are
7.4；Preparation method such as 1,121 DEG C of steam sterilizing 20min of embodiment.
2nd, the strain passage culture of various concentrations culture medium
Above-mentioned bacillus subtilis ANSB471 is taken to be inoculated into the fermentation of 50ml original concentrations with the inoculum concentration of 2% (volume ratio)
In the 250ml triangular flasks of liquid ((fermentation medium of above-mentioned " 1, the preparation of seed liquor in ")), fermentation temperature is 37 DEG C, pH value
7.0, rotating speed 200r/min, fermentation time 36h, obtains zymotic fluid A.
Above-mentioned bacillus subtilis ANSB471 zymotic fluids A is taken, after 80 DEG C of heating water bath 15min, with 2% (volume ratio)
Inoculum concentration is inoculated into the 250ml triangular flasks of 50% original concentration zymotic fluids of 50ml, and fermentation temperature is 37 DEG C, and pH value 7.0, turns
Fast 200r/min, fermentation time 36h, obtains zymotic fluid B.The 50% concentration cultures concentration is the 1/ of original culture medium concentration
2,121 DEG C of steam sterilizing 20min.
Above-mentioned bacillus subtilis ANSB471 zymotic fluids B is taken, after 80 DEG C of heating water bath 15min, with 2% (volume ratio)
Inoculum concentration is inoculated into the 250ml triangular flasks of 12.5% original concentration zymotic fluids of 50ml, and fermentation temperature is 37 DEG C, pH value 7.0,
Rotating speed 200r/min, fermentation time 36h, obtains zymotic fluid C.The 12.5% concentration cultures concentration is original culture medium concentration
1/8,121 DEG C of steam sterilizing 20min.
According to above-mentioned fermented and cultured mode, the secondary culture on 50% concentration cultures, original culture medium successively.Per generation
Zymotic fluid is used as follow-on seed liquor after 80 DEG C of heating water bath 15min.Trained successively in original culture medium, 50% concentration
Support base, 12.5% concentration cultures, 50% concentration cultures, 5 generation of secondary culture on original culture medium.
3rd, the calculating of gemma rate
Often it is commissioned to train after supporting, using the method for plate culture count detection bacterium sum；By 80 DEG C of heating water bath 15min
Afterwards, using the growing amount of the method for plate culture count detection gemma.The calculation of wherein gemma rate is as follows：
Gemma rate (%)=(gemma number/total viable count) * 100%.
Total viable count and gemma detection are nutrient agar (NA) culture medium with culture medium, and specific component is (g/L)：Peptone
10g, beef extract 3g, sodium chloride 5g, agar 15g.
After 5 are commissioned to train and support, the production gemma rate of bacillus subtilis ANSB471 has brought up to 93% from 81%.
Claims (10)
- A kind of 1. strain domestication method for improving bacillus production gemma rate, it is characterised in that comprise the following steps：Bacillus is activated；By the strain after activation successively according to from normal incubation medium to barren culture medium, nutrient concentrations are then further added by just The order cultivated in normal culture medium carries out secondary culture, and culture medium sets more than 3 according to concentration, and continuous passage 3 is more than generation.
- 2. strain domestication method according to claim 1, it is characterised in that the normal incubation medium is original for 80-100% The culture medium of concentration, the barren culture medium are 12.5%-80% original concentration culture mediums.
- 3. the strain domestication method according to right wants 1 or 2, it is characterised in that described from normal incubation medium to barren culture Base arrives normal incubation medium again, 3-9 concentration gradient is progressively set, from normal incubation medium to barren culture medium, before latter concentration is The 12.5%-80% of one concentration；From barren culture medium to normal incubation medium, previous concentration is the 12.5%-80% of latter concentration.
- 4. strain domestication method according to claim 3, it is characterised in that described from normal incubation medium to barren culture medium Normal incubation medium is arrived again, 3-7 concentration gradient is progressively set, and from normal incubation medium to barren culture medium, latter concentration is previous The 12.5%-80% of concentration；From barren culture medium to normal incubation medium, previous concentration is the 12.5%-80% of latter concentration.
- 5. strain domestication method according to claim 4, it is characterised in that described from normal incubation medium to barren culture medium The order for arriving normal incubation medium again is：Original concentration culture medium, 50% original concentration culture medium, 25% original concentration culture medium, 12.5% original concentration culture medium, 25% original concentration culture medium, 50% original concentration culture medium, original concentration culture medium.
- 6. according to claim 2-5 any one of them strain domestication methods, it is characterised in that the original concentration culture medium For：Per 1000mL, following component is included：Tryptone 8-12g, yeast extract 1.5-3g, glucose 1.5-4g, beef extract 2- 5g, sodium chloride 3-6g, disodium hydrogen phosphate 2.5-4g, epsom salt 0.5-1.5g, pH value 7.0-7.4.
- 7. according to claim 1-6 any one of them strain domestication methods, it is characterised in that after every generation terminates culture, Heating water bath, is then passed on next time again.
- 8. strain domestication method according to claim 7, it is characterised in that the method for the heating water bath is：80-100 DEG C, heat 5-20min.
- 9. strain domestication method according to claim 1, it is characterised in that the condition of the secondary culture is：Shaking flask is sent out Ferment, fermentation temperature are 30-37 DEG C, fermentation time 30-48h, rotating speed 180-220r/min, pH value 7.0-7.4, preferably , fermentation temperature is 37 DEG C, fermentation time 36h, rotating speed 200r/min, pH value 7.0.
- 10. strain domestication method according to claim 1, it is characterised in that the bacillus is that can form gemma Bacillus or coccus, including bacillus, Sporolactobacillus, fusobacterium, Desulfotomaculum and Sporosarcina, it is excellent Choosing, be bacillus subtilis, bacillus licheniformis, bacillus megaterium, bacillus coagulans, bacillus cereus, natto Bacillus or clostridium butyricum.
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| CN113215056A (en) * | 2021-06-02 | 2021-08-06 | 亚太星原农牧科技海安有限公司 | Screening method of bacillus coagulans capable of easily forming spores |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215056A (en) * | 2021-06-02 | 2021-08-06 | 亚太星原农牧科技海安有限公司 | Screening method of bacillus coagulans capable of easily forming spores |
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