CN108795792A - A kind of culture medium and fermentation process of bacillus licheniformis - Google Patents
A kind of culture medium and fermentation process of bacillus licheniformis Download PDFInfo
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Abstract
The invention discloses a kind of culture medium of bacillus licheniformis and fermentation process, contain phosphate and manganese salt in culture medium, it can promote bacillus licheniformis fast-growth, bacillus is set to rapidly enter exponential phase, the thalli growth phase can be extended simultaneously, to greatly improve bacillus licheniformis product yield.The fermentation process of the culture medium is activation, the culture of level-one shake-flask seed and the ferment tank culture of seed.
Description
Technical field
The present invention relates to a kind of culture medium of bacillus licheniformis and fermentation process.
Background technology
Bacillus licheniformis (Bacillus licheniformis) is a kind of bacterial strain having specific function comprising many,
Have a wide range of applications in the scientific domains research such as industry, agricultural, medicine.The zymotechnique of bacillus licheniformis is universal at present
The defects of it is relatively low that there is fermentation levels, and sporulation is inconsistent, product quantity and unstable quality.
The prior art is in order to improve the fermentation level of bacillus licheniformis, on the one hand, by during fermented and cultured to hair
The adjustment of ferment technique such as adjusts fermentation temperature, pressure, time process conditions, to improve bacillus licheniformis yield, but it is real
Border operates and very bothers, while increasing production cost, can not greatly improve yield；On the other hand, by lichens
Bacillus culture medium prescription is adjusted, and the main component of the culture medium prescription of existing general production bacillus licheniformis is
The selection of nitrogen source, carbon source, inorganic salts etc., wherein inorganic salts is more, such as sodium salt, sylvite, molysite, magnesium salts and calcium salt etc..It is certain
A small amount of phosphate or manganese salt are added in ground bacillus culture medium, but bacillus growth is not obvious,《Micro- Mn
Ion pair DY bacillus spores form the research influenced》(Chinese microecology magazine the 12nd phase of volume 25 in December, 2013) is public
Spore forming rate during DY fermentation of bacillus can be obviously improved by having opened manganese ion, influence thalli growth curve, promote gemma
Formation, make microbial activity higher, to make ferment effect be significantly improved.Related phosphate promotes bacillus licheniformis
The document of fast-growth has not been reported.
Invention content
The technical problem to be solved in the present invention is to provide a kind of bacillus licheniformis culture medium, which can promote lichens
Bacillus fast-growth makes bacillus rapidly enter exponential phase, while can extend the thalli growth phase, to carry significantly
The yield of high bacillus.
The present invention solves above-mentioned technical problem using following technical scheme.
The technical scheme is that：
A kind of bacillus licheniformis culture medium, the culture medium phosphate-containing and manganese salt.
Bacillus licheniformis culture medium main component provided by the invention is that this field is conventional, and main component further includes：
Nitrogen source, carbon source and sodium chloride.
Bacillus licheniformis culture medium main component provided by the invention is that this field is conventional.Nitrogen source is selected from peptone, flower
It is one or more in raw cake powder, soybean cake powder, cotton seed powder cake, corn steep liquor, yeast powder, fish meal, dried silkworm chrysalis meal and urea, preferred egg
White peptone.Carbon source is one or more in beef extract, cornstarch, corn steep liquor, lactose, sucrose and glucose, preferably beef
Cream.The one kind of phosphate in ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate
Or it is a variety of.Manganese salt can be selected from manganese chloride or manganese sulfate.
Nitrogen source is that this field is conventional in the present invention, and the content range of the nitrogen source is preferably 5~30g/L.
Carbon source is that this field is conventional in the present invention, and the content range of the carbon source is preferably 3~7g/L.
Sodium chloride is that this field is conventional in the present invention, and the content range of the sodium chloride is preferably 5~15g/L.
Phosphate is that this field is conventional in the present invention, and the phosphatic content range is preferably 0.1~6g/L.
Manganese salt is that this field is conventional in the present invention, and the content range of the manganese salt is preferably 0.03~10.0g/L.
Preferably, the main component of the culture medium includes：5~30g/L of peptone, 3~7g/L of beef extract, sodium chloride 5
~15g/L, phosphate 0.1g/L~6.0g/L and manganese salt 0.03mg/L~10.0mg/L.
More preferably, the main component of the culture medium includes：7~12g/L of peptone, 4~6g/L of beef extract, sodium chloride 7
0.03~3.0mg/L of~13g/L, 1.0~3.0g/L of phosphate and manganese salt.
In the preferred embodiment of the present invention, the culture medium is only by 5~30g/L of peptone, 3~7g/ of beef extract
L, 5~15g/L of sodium chloride, phosphate 0.1g/L~6.0g/L and manganese salt 0.03mg/L~10.0mg/L compositions；More preferably, described
Culture medium is only by 7~12g/L of peptone, 4~6g/L of beef extract, 7~13g/L of sodium chloride, 1.0~3.0g/L of phosphate and manganese salt
0.03~3.0mg/L is formed.
The fermentation culture method of bacillus licheniformis provided by the invention is that this field is conventional, the main work for including strain
Change, level-one shaking flask Spawn incubation and fermented and cultured and etc..Fermented and cultured concrete operation step is as follows：
1) activation of seed：Shaking flask meat soup culture is added in bacillus licheniformis seed；
2) level-one shake-flask seed culture：The seed access level-one Shake flask medium of the activation of certain inoculum concentration is further trained
It supports；
3) fermented and cultured：The first order seed access of certain inoculum concentration is subjected to fermented and cultured.
The condition of bacillus licheniformis seed activation of the present invention can be it is conventional, preferably, temperature be 30~
38 DEG C, rotating speed is 120~200r/min, and the time is 6~8h.
The inoculum concentration of Oscillating bottles of seeds of Yi Ji of the present invention can be it is conventional, preferable inoculum concentration be 0.5%~
5%, the percentage is percent by volume.The condition of level-one shake-flask seed culture can make it is conventional, preferably, temperature be 30
~38 DEG C, rotating speed is 120~200r/min, and shaking flask incubation time is 6~8h.
The inoculum concentration of fermented and cultured of the present invention can be conventional, and preferable inoculum concentration is 20~35%, described
Percentage is percent by volume.The conditional parameter of fermented and cultured can be conventional, preferably, fermentation tankage size is 15L, fermentation
The liquid amount of liquid is 50~60%, and the percentage is percent by volume, and fermentation temperature is 30~38 DEG C, and tank pressure is 0.03Mpa,
Ventilating ratio is 1:0.5~1, dissolved oxygen amount is 20~40%, and the percentage is the percentage of relative saturation dissolved oxygen, fermentation time
For for 24 hours.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined arbitrarily to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The beneficial effects of the present invention are：
Bacillus licheniformis medium component source be easy to get and it is cheap, technological parameter is simple, add simultaneously in culture medium
It phosphorates hydrochlorate and manganese salt can promote bacillus licheniformis fast-growth.Bacillus is set to rapidly enter exponential phase, while energy
Extend the thalli growth phase, to greatly improve the yield of bacillus.
Specific implementation mode
It is further illustrated the present invention below with embodiment, but the present invention is not intended to be limited thereto.It is not noted in the following example
The experimental method of bright actual conditions, usually according to normal condition.
In following embodiments, the bacillus licheniformis seed source Zhejiang Jingxin Pharmaceutical Co., Ltd used, lot number
20161115-25.Water used in fermentation medium is purified water.
Comparative example 1：Blank control group
After the purifying water dissolution of ampoul tube seed, it is added to shaking flask meat soup culture, the bottled liquid 150ml of 500ml triangles,
37.5 DEG C, shaking flask activates 6h under conditions of 180r/min；The seed access level-one shaking flask training for activating shaking flask by 0.1% inoculum concentration
Foster base, the bottled liquid 300ml of 1000ml triangles, at 37.5 DEG C, shaking flask culture 6h under conditions of 180r/min.Level-one Shake flask medium
Ingredient include peptone 10g/L, beef extract 5g/L and sodium chloride 10g/L；First order seed is transferred to according to 30% inoculum concentration
15 liters of fermentation tanks ferment, and ferment liquid amount 60%.In 37.5 DEG C of fermentation, tank presses 0.03Mpa, ventilating ratio 1:0.8, dissolved oxygen amount
It ferments for 24 hours under the conditions of 30%.The ingredient of fermentation medium includes peptone 10g/L, beef extract 5g/L and sodium chloride 10g/L.Hair
For zymotic fluid after centrifugation, thalline weight in wet base is 4.0g/L, 30% or less gemma rate.
Comparative example 2：Manganese salt 0.03mg/L
After the purifying water dissolution of ampoul tube seed, it is added to shaking flask meat soup culture, the bottled liquid 150ml of 500ml triangles, 34
DEG C, shaking flask activates 6h under conditions of 180r/min；Level-one shaking flask culture is accessed by the seed that 0.5% inoculum concentration activates shaking flask
Base, the bottled liquid 300ml of 1000ml triangles, at 34 DEG C, shaking flask culture 6h under conditions of 200r/min.Level-one Shake flask medium at
It includes peptone 10g/L, beef extract 5g/L and sodium chloride 10g/L to divide；First order seed is transferred to 15 liters according to 20% inoculum concentration
Fermentation tank ferments, and ferment liquid amount 60%.In 34 DEG C of fermentation, tank presses 0.03Mpa, ventilating ratio 1:0.8, dissolved oxygen amount 20%
It ferments for 24 hours under part.The ingredient of fermentation medium includes peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and a Heshui sulphur
Sour manganese 0.03mg/L.For zymotic fluid after centrifugation, thalline weight in wet base is 4.2g/L, 40% or less gemma rate.
Comparative example 3：Manganese salt 0.3mg/L
The fermentation culture conditions parameter of comparative example 3 is just the same with comparative example 2, differs only in：Fermentation training
The ingredient for supporting base includes peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and a Heshui manganese sulfate 0.3mg/L.Zymotic fluid
After centrifugation, thalline weight in wet base is 4.7g/L, 60% or less gemma rate.
Comparative example 4：Manganese salt 3.0mg/L
The fermentation culture conditions parameter of comparative example 4 is just the same with comparative example 2, differs only in：Fermentation training
The ingredient for supporting base includes peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and a Heshui manganese sulfate 3.0mg/L.Zymotic fluid
After centrifugation, thalline weight in wet base is 4.0g/L, 70% or less gemma rate.
Comparative example 5：Manganese salt 10.0mg/L
The fermentation culture conditions parameter of comparative example 5 is just the same with comparative example 2, differs only in：Fermentation training
The ingredient for supporting base includes peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and a Heshui manganese sulfate 10.0mg/L.Zymotic fluid
After centrifugation, thalline weight in wet base is 3.2g/L, 60% or less gemma rate.
Comparative example 6:Phosphate 0.1g/L
After the purifying water dissolution of ampoul tube seed, it is added to shaking flask meat soup culture, the bottled liquid 150ml of 500ml triangles, 30
DEG C, shaking flask activates 7h under conditions of 150r/min；Level-one Shake flask medium is accessed by the seed that 1% inoculum concentration activates shaking flask,
The bottled liquid 300ml of 1000ml triangles, at 30 DEG C, shaking flask culture 7h under conditions of 150r/min.The ingredient of level-one Shake flask medium
Including peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and purified water.First order seed is transferred to according to 30% inoculum concentration
15 liters of fermentation tanks ferment, and ferment liquid amount 60%.In 30 DEG C of fermentation temperature, tank presses 0.03Mpa, ventilating ratio 1:0.6, dissolved oxygen
It ferments for 24 hours under the conditions of amount 30%.The ingredient of fermentation medium include peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and
Dipotassium hydrogen phosphate 0.1g/L.For zymotic fluid after centrifugation, thalline weight in wet base is 6.2g/L, 30% or less gemma rate.
Comparative example 7:Phosphate 1.0g/L
The fermentation culture conditions parameter of comparative example 7 is just the same with comparative example 6, differs only in：Fermentation training
The ingredient for supporting base includes peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and dipotassium hydrogen phosphate 1.0g/L.Zymotic fluid pass through from
After the heart, thalline weight in wet base is 7.3g/L, 30% or less gemma rate.
Comparative example 8:Phosphate 3.0g/L
The fermentation culture conditions parameter of comparative example 8 is just the same with comparative example 6, differs only in：Fermentation training
The ingredient for supporting base includes peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and dipotassium hydrogen phosphate 3.0g/L.Zymotic fluid pass through from
After the heart, thalline weight in wet base is 7.8g/L, 20% or less gemma rate.
Comparative example 9:Phosphate 6.0g/L
The fermentation culture conditions parameter of comparative example 9 is just the same with comparative example 6, differs only in：Fermentation training
The ingredient for supporting base includes peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L and dipotassium hydrogen phosphate 6.0g/L.Zymotic fluid pass through from
After the heart, thalline weight in wet base is 7.1g/L, 20% or less gemma rate.
Comparative example 1-9 test results are as shown in the table
Embodiment 1：Phosphate:0.1g/L, manganese salt 0.3mg/L
The fermentation culture conditions parameter of embodiment 1 is just the same with comparative example 2, differs only in：Fermentation medium
Ingredient include peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L, dipotassium hydrogen phosphate 0.1g/L and a Heshui manganese sulfate
0.3mg/L.For zymotic fluid after centrifugation, thalline weight in wet base is 7.5g/L, 80% or more gemma rate.
Embodiment 2:Phosphate 1.0g/L, manganese salt 0.3mg/L
The fermentation culture conditions parameter of embodiment 2 is just the same with comparative example 2, differs only in：Fermentation medium
Ingredient include peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L, dipotassium hydrogen phosphate 1.0g/L and a Heshui manganese sulfate
0.3mg/L.For zymotic fluid after centrifugation, thalline weight in wet base is 7.0g/L, 70% or more gemma rate.
Embodiment 3:Phosphate 1.0g/L, manganese salt 3.0mg/L
The fermentation culture conditions parameter of embodiment 3 is just the same with comparative example 2, differs only in：Fermentation medium
Ingredient include peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L, dipotassium hydrogen phosphate 1.0g/L and a Heshui manganese sulfate
3.0mg/L.For zymotic fluid after centrifugation, thalline weight in wet base is 8.0g/L, 85% or more gemma rate.
Embodiment 4:Phosphate 3.0g/L, manganese salt 0.03mg/L
The fermentation culture conditions parameter of embodiment 4 is just the same with comparative example 2, differs only in：Fermentation medium
Ingredient include peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L, dipotassium hydrogen phosphate 3.0g/L and a Heshui manganese sulfate
0.03mg/L.For zymotic fluid after centrifugation, thalline weight in wet base is 8.5g/L, 80% or more gemma rate.
Embodiment 5:Phosphate 3.0g/L, manganese salt 0.3mg/L
The fermentation culture conditions parameter of embodiment 5 is just the same with comparative example 2, differs only in：Fermentation medium
Ingredient include peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L, dipotassium hydrogen phosphate 3.0g/L and a Heshui manganese sulfate
0.3mg/L.For zymotic fluid after centrifugation, thalline weight in wet base is 9.2g/L, 90% or more gemma rate.
Embodiment 6：Phosphate:6.0g/L, manganese salt 10.0mg/L
The fermentation culture conditions parameter of embodiment 6 is just the same with comparative example 2, differs only in：Fermentation medium
Ingredient include peptone 10g/L, beef extract 5g/L, sodium chloride 10g/L, dipotassium hydrogen phosphate 6.0g/L and a Heshui manganese sulfate
10.0mg/L.For zymotic fluid after centrifugation, thalline weight in wet base is 7.3g/L, 70% or more gemma rate.
Embodiment 1-6 group test results are as shown in the table：
Conclusion：
It is compared from above-mentioned 6 groups of embodiments and 10 groups of comparative examples, it can be seen that when culture medium phosphate-containing and manganese salt,
Bacillus licheniformis growth result is apparently higher than growth result of the culture medium containing only phosphate or manganese salt when；Further, from implementation
Example 1-6 can be seen that, when phosphate concn is 3.0g/L, when a concentration of 0.3mg/L of manganese salt, the equal thalline weight in wet base of bacillus licheniformis and
Gemma rate highest, bacillus licheniformis growth-promoting effect is best at this time.
Claims (10)
1. a kind of bacillus licheniformis culture medium, which is characterized in that phosphate-containing and manganese salt in culture medium.
2. culture medium according to claim 1, which is characterized in that its main component further includes：Nitrogen source, carbon source and chlorination
Sodium.
3. culture medium according to claim 2, which is characterized in that nitrogen source be selected from peptone, groundnut meal, soybean cake powder,
It is one or more in cotton seed powder cake, corn steep liquor, yeast powder, fish meal, dried silkworm chrysalis meal and urea, optimization protein peptone；
And/or carbon source is one or more in beef extract, cornstarch, corn steep liquor, lactose, sucrose and glucose, preferably
Beef extract；
And/or phosphate is in ammonium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and sodium dihydrogen phosphate
It is one or more；
And/or manganese salt is selected from manganese chloride or manganese sulfate.
4. culture medium according to claim 2, which is characterized in that the content of the nitrogen source is 5~30g/L.
And/or the content range of the carbon source is 3~7g/L.
And/or the content range of the inorganic salts is 5~15g/L.
And/or the phosphatic content range is 0.1~6.0g/L.
And/or the content range of the manganese salt is 0.03~10.0mg/L.
5. culture medium according to claim 1, which is characterized in that the main component of the culture medium includes：Peptone 5~
30g/L, 3~7g/L of beef extract, 5~15g/L of sodium chloride, phosphate 0.1g/L~6.0g/L and manganese salt 0.03mg/L~
10.0mg/L。
6. culture medium according to claim 5, which is characterized in that the main component of the culture medium includes：Peptone 7~
12g/L, 4~6g/L of beef extract, 0.03~3.0mg/L of 7~13g/L of sodium chloride, 1.0~3.0g/L of phosphate and manganese salt.
7. a kind of the lichen bacillus ferments cultural method, which is characterized in that fermentation process is as follows：
1) activation of seed：Shaking flask meat soup culture is added in bacillus licheniformis seed；
2) level-one shake-flask seed culture：The activated seed access level-one Shake flask medium of certain inoculum concentration is further cultivated；
3) ferment tank culture：Culture described in any one of first order seed access claim 1-6 by certain inoculum concentration
Base carries out fermented and cultured.
8. fermentation culture method according to claim 7, which is characterized in that the activation and level-one shake-flask seed culture of seed
Conditional parameter is：Temperature is 30~38 DEG C, and rotating speed is 120~200r/min, and the time is 6~8h.
9. fermentation culture method according to claim 7, which is characterized in that fermentation culture conditions are：Fermentation liquid amount be
50~60%, the percentage is percent by volume, and temperature is 30~38 DEG C, and tank pressure is 0.03Mpa, ventilating ratio 1:0.5~
1, dissolved oxygen amount is 20~40%, and the percentage is the percentage of relative saturation dissolved oxygen, and fermentation time is for 24 hours.
10. fermentation culture method according to claim 7, which is characterized in that the inoculum concentration of Oscillating bottles of seeds of Yi Ji be 0.5~
5%, the inoculum concentration of fermented and cultured is 20~35%, and the percentage is percent by volume.
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Application publication date: 20181113 |