CN109837320A - A method of it improving denitrified pseudomonas and produces vitamin B12 - Google Patents
A method of it improving denitrified pseudomonas and produces vitamin B12 Download PDFInfo
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- CN109837320A CN109837320A CN201910238608.0A CN201910238608A CN109837320A CN 109837320 A CN109837320 A CN 109837320A CN 201910238608 A CN201910238608 A CN 201910238608A CN 109837320 A CN109837320 A CN 109837320A
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Abstract
The present invention relates to technical field of biological fermentation, especially a kind of method for improving denitrified pseudomonas and producing vitamin B12.The method that a kind of raising denitrified pseudomonas of the invention produces vitamin B12 cultivates the production bacterial strain of vitamin B12 by female bottle culture medium, Shake flask medium and the supplemented medium optimized through many experiments under optimal fermentation condition；Control temperature is in a relatively low level in the 0-39h of fermentation process early period, i.e., and 26-30 DEG C, so that controlling strain be in suitable growth rate and the good bacterium shape of holding；The later period improves and maintains relatively high level of the fermentation system temperature at one during the fermentation, i.e. 30-34 DEG C, to promote the starting and accumulation of thalli growth, vitamin B12 synthesis, and then make after fermentation, vitamin B12 fermentation unit is up to 198mg/L, it not only increases the yield and production efficiency of vitamin B12, and energy saving, reduces cost.
Description
Technical field
The present invention relates to technical field of biological fermentation, especially a kind of denitrified pseudomonas that improves produces vitamin B12
Method.
Background technique
Vitamin B12 (Vitamin B12) is also known as cobalamin, is a kind of corrin class compound general name containing cobalt, earliest
It is found as " antipernicious anemia factor " in nineteen twenty-six.Vitamin B12 is a kind of important bioactive substance, is also simultaneously
The growth factor of many microorganisms and animal is widely used in medicine, food and animal husbandry.Due to the molecule knot of vitamin B12
Structure is extremely complex, and full chemistry synthesis needs up to more than 70 reaction steps, and expensive, therefore is manufactured almost exclusively by present
The mode of microbial fermentation produces vitamin B12.In nature, there are two different ways for the biosynthesis of vitamin B12
Diameter, i.e., aerobic approach and anaerobic pathway, but the biosynthesis of VB12 is limited strictly in some microorganisms, is applied successfully at present
Mainly there is propionibacterium freudenreichii (Propiomibacterium freudenreichii) in the production bacterial strain of industrial fermentation, thank
Family name's Propionibacterium (Propiomibacterium shermanii) and denitrified pseudomonas (Pseudomonas
Denitrificans).Wherein, because the approach of the aerobic synthesis vitamin B12 of denitrified pseudomonas is clear and vitamin
B12 yield is high, thus, industrial production is nearly all the work using denitrified pseudomonas as production vitamin B12 at present
Industry bacterial strain.
The Process control conditions that denitrified pseudomonas aerobic fermentation produces vitamin B12 are stringenter, once control is not
It will lead to the apprehensive early ageing of thallus well and then influence the yield of vitamin B12.Therefore, denitrogenation vacation unit cell bar is maintained in fermentation process
The thalli morphology of bacterium is most important.Other than fermentation medium, pH and revolving speed etc. can influence thalli morphology, temperature is also shadow
Ring the key factor of strain bacterium shape.For same microorganism, different physiological and biochemical procedures has different optimum temperatures,
That is cultivation temperature or accumulation metabolite when cultivation temperature when thalli growth amount highest is not equal to fermentation rate highest
Measure cultivation temperature when highest.Therefore, thalli growth is conducive to reach earlier fermentation to adjustment temperature in fermentation process,
Middle and later periods is conducive to the purpose of secondary metabolite accumulation, and then promotes vitamin B12 yield.
Since vitamin B12 application field is more and more wider, currently, vitamin B12 demand is larger, therefore this field is urgent
The method for needing to develop efficient production vitamin B12, will not only improve the yield of vitamin B12, but also save as far as possible
Energy consumption reduces cost, production efficiency is improved, to meet the needs of market.
Summary of the invention
The purpose of the present invention is to provide a kind of methods that raising denitrified pseudomonas produces vitamin B12, improve dimension life
The yield of plain B12, energy saving reduce cost, improve production efficiency.
The technical scheme adopted by the invention to solve the technical problem is that:
A method of it improving denitrified pseudomonas and produces vitamin B12, characterized by the following steps:
S1, female bottle incubation control: the denitrified pseudomonas two generations inclined-plane of activation is scraped into appropriate thallus and is inoculated in female bottle
In culture medium, temperature control is at 27-29 DEG C, and for humid control 30-60%, incubation time was 18-hours for 24 hours；
S2, the control of shaking flask incubation: standard compliant denitrified pseudomonas mother bottle culture medium is aseptically accessed
Shake flask medium, inoculum concentration 10%, earlier fermentation (0-39h) shaking table temperature are controlled at 26-30 DEG C；Ferment middle and later periods (39-
170h) control of shaking table temperature is at 30-34 DEG C, and humid control is 30-60%；Incubation time is 170h；
S3, control of additive raw material: in the fermentation process of step S2, total sugar content is detected when 3d, 4d, 5d, works as total sugar content
Feed supplement is carried out lower than 5%, maintains the control of fermentation liquid total sugar content in 5-7%, until fermentation ends, isolate and purify out from fermentation liquid and tie up
Raw element B12.
Preferably, the preparation steps of female bottle culture medium include: to weigh beet molasses (50%) respectively in the step S1
110g, dipotassium hydrogen phosphate 0.60g, ammonium sulfate 0.81g, magnesium sulfate 1.65g, manganese sulfate 0.15g, zinc sulfate 0.02g, cobalt chloride
0.02g, precursor 0.012g, dissolve and are settled to 1000mL, adjust pH, 6.80-7.40 before disappearing with 20% NaOH solution, each
300mL triangular flask dispenses 60mL, and female bottle culture medium is made after sterilized (0.11-0.12MPa, 121 DEG C, 30min) in wrapping.
Preferably, the preparation steps of Shake flask medium include: to weigh beet molasses (50%) respectively suddenly in the step S2
170g, sucrose 15g, choline chloride 10.30g, ammonium sulfate 2.60g, magnesium sulfate 1.65g, zinc sulfate 0.08g, cobalt chloride 0.14g,
Precursor 0.01g, glycerol 1.55g, phosphoglycerol 7g, dissolve and are settled to 1000mL, with 20% NaOH solution tune pH, before disappearing
6.80-7.40, each 300mL triangular flask dispense 37mL, and wrapping is made after sterilized (0.11-0.12MPa, 121 DEG C, 30min)
Shake flask medium.
Preferably, the preparation steps of supplemented medium include: to weigh sucrose 300g, DMBI respectively in the step S3
0.20g, CoCl2·6H2O 0.23g dissolves and is settled to 1000mL, adjusts pH to 6.80-7.40, sterilized (0.11-
0.12MPa, 121 DEG C, 30min) after supplemented medium is made.
Preferably, in the step S2 standard compliant denitrified pseudomonas mother bottle culture medium feature are as follows: without miscellaneous bacteria,
OD value is 0.207-0.220.
The beneficial effects of the present invention are: compared with prior art, a kind of raising denitrified pseudomonas of the invention produces dimension
The method of raw element B12, by female bottle culture medium, Shake flask medium and the supplemented medium optimized through many experiments, in optimal hair
The production bacterial strain of vitamin B12 is cultivated under the conditions of ferment；Control temperature is relatively low at one in the 0-39h of fermentation process early period
Level, i.e., 26-30 DEG C are in suitable growth rate and keep good bacterium shape to control strain；After during the fermentation
Relatively high level of the fermentation system temperature at one is improved and maintained to phase, i.e., and 30-34 DEG C, to promote thalli growth, dimension life
The starting and accumulation of plain B12 synthesis, and then make after fermentation, vitamin B12 fermentation unit is up to 198mg/L, not only improves
The yield and production efficiency of vitamin B12, and energy saving, reduce cost.
Specific embodiment
A kind of method for improving denitrified pseudomonas and producing vitamin B12 of embodiment 1
It is a kind of improve denitrified pseudomonas produce vitamin B12 method include the following steps:
S1, female bottle incubation control: the denitrified pseudomonas two generations inclined-plane of activation is scraped into appropriate thallus and is inoculated in female bottle
In culture medium, temperature control is at 27 DEG C, and for humid control in 30-35%, incubation time is 18h hours；Female bottle culture medium OD at this time:
0.220；PH:7.34；
S2, the control of shaking flask incubation: by standard compliant denitrified pseudomonas mother bottle culture medium, (no miscellaneous bacteria, OD value are
0.220) Shake flask medium, inoculum concentration 10% are aseptically accessed, earlier fermentation (0-35h) shaking table temperature is controlled 27
±0.5℃；Middle and later periods (35-167h) shaking table temperature of fermenting control is at 33 ± 0.5 DEG C, and humid control is in 30-35%；Incubation time
For 167h；
S3, control of additive raw material: in the fermentation process of step S2, total sugar content is detected when 3d, 4d, 5d, works as total sugar content
Feed supplement is carried out lower than 5%, maintains the control of fermentation liquid total sugar content in 5-7%, until fermentation ends, isolate and purify out from fermentation liquid and tie up
Raw element B12.
In the present embodiment, the preparation steps of female bottle culture medium include: to weigh beet molasses respectively in the step S1
(50%) 110g, dipotassium hydrogen phosphate 0.60g, ammonium sulfate 0.81g, magnesium sulfate 1.65g, manganese sulfate 0.15g, zinc sulfate 0.02g, chlorine
Change cobalt 0.02g, precursor 0.012g, dissolve and be settled to 1000mL, adjusts pH, 6.80-7.40 before disappearing with 20% NaOH solution,
Each 300mL triangular flask dispenses 60mL, and female bottle culture is made after sterilized (0.11-0.12MPa, 121 DEG C, 30min) in wrapping
Base.
In the present embodiment, the preparation steps of Shake flask medium include: to weigh beet molasses respectively suddenly in the step S2
(50%) 170g, sucrose 15g, choline chloride 10.30g, ammonium sulfate 2.60g, magnesium sulfate 1.65g, zinc sulfate 0.08g, cobalt chloride
0.14g, precursor 0.01g, glycerol 1.55g, phosphoglycerol 7g, dissolve and are settled to 1000mL, with 20% NaOH solution tune pH,
6.80-7.40 before disappearing, each 300mL triangular flask dispense 37mL, wrapping, after sterilized (0.11-0.12MPa, 121 DEG C, 30min)
Shake flask medium is made.
In the present embodiment, the preparation steps of supplemented medium include: to weigh sucrose 300g respectively in the step S3,
DMBI 0.20g, CoCl2·6H2O 0.23g dissolves and is settled to 1000mL, adjusts pH to 6.80-7.40, sterilized (0.11-
0.12MPa, 121 DEG C, 30min) after supplemented medium is made.
Through detecting, fermentation ends, vitamin B12 fermentation unit is 189mg/L.
A kind of method for improving denitrified pseudomonas and producing vitamin B12 of embodiment 2
It is a kind of improve denitrified pseudomonas produce vitamin B12 method include the following steps:
S1, female bottle incubation control: the denitrified pseudomonas two generations inclined-plane of activation is scraped into appropriate thallus and is inoculated in female bottle
In culture medium, temperature control is at 29 DEG C, and for humid control in 50-60%, incubation time is hour for 24 hours；Female bottle culture medium OD at this time:
0.212；PH:7.42；
S2, the control of shaking flask incubation: by standard compliant denitrified pseudomonas mother bottle culture medium, (no miscellaneous bacteria, OD value are
0.212) Shake flask medium, inoculum concentration 10% are aseptically accessed, earlier fermentation (0-38h) shaking table temperature is controlled 29
±0.5℃；Middle and later periods (38-169h) shaking table temperature of fermenting control is at 32 ± 0.5 DEG C, and humid control is in 50-60%；Incubation time
For 169h；
S3, control of additive raw material: in the fermentation process of step S2, total sugar content is detected when 3d, 4d, 5d, works as total sugar content
Feed supplement is carried out lower than 5%, maintains the control of fermentation liquid total sugar content in 5-7%, until fermentation ends, isolate and purify out from fermentation liquid and tie up
Raw element B12.
In the present embodiment, the preparation steps of female bottle culture medium include: to weigh beet molasses respectively in the step S1
(50%) 110g, dipotassium hydrogen phosphate 0.60g, ammonium sulfate 0.81g, magnesium sulfate 1.65g, manganese sulfate 0.15g, zinc sulfate 0.02g, chlorine
Change cobalt 0.02g, precursor 0.012g, dissolve and be settled to 1000mL, adjusts pH, 6.80-7.40 before disappearing with 20% NaOH solution,
Each 300mL triangular flask dispenses 60mL, and female bottle culture is made after sterilized (0.11-0.12MPa, 121 DEG C, 30min) in wrapping
Base.
In the present embodiment, the preparation steps of Shake flask medium include: to weigh beet molasses respectively suddenly in the step S2
(50%) 170g, sucrose 15g, choline chloride 10.30g, ammonium sulfate 2.60g, magnesium sulfate 1.65g, zinc sulfate 0.08g, cobalt chloride
0.14g, precursor 0.01g, glycerol 1.55g, phosphoglycerol 7g, dissolve and are settled to 1000mL, with 20% NaOH solution tune pH,
6.80-7.40 before disappearing, each 300mL triangular flask dispense 37mL, wrapping, after sterilized (0.11-0.12MPa, 121 DEG C, 30min)
Shake flask medium is made.
In the present embodiment, the preparation steps of supplemented medium include: to weigh sucrose 300g respectively in the step S3,
DMBI 0.20g, CoCl2·6H2O 0.23g dissolves and is settled to 1000mL, adjusts pH to 6.80-7.40, sterilized (0.11-
0.12MPa, 121 DEG C, 30min) after supplemented medium is made.
Through detecting, fermentation ends, vitamin B12 fermentation unit is 195 mg/L.
A kind of method for improving denitrified pseudomonas and producing vitamin B12 of embodiment 3
It is a kind of improve denitrified pseudomonas produce vitamin B12 method include the following steps:
S1, female bottle incubation control: the denitrified pseudomonas two generations inclined-plane of activation is scraped into appropriate thallus and is inoculated in female bottle
In culture medium, temperature control is at 28 DEG C, and for humid control in 10-50%, incubation time is 22h hours；Female bottle culture medium OD at this time:
0.207；PH:7.39；
S2, the control of shaking flask incubation: by standard compliant denitrified pseudomonas mother bottle culture medium, (no miscellaneous bacteria, OD value are
0.207) Shake flask medium, inoculum concentration 10% are aseptically accessed, earlier fermentation (0-39h) shaking table temperature is controlled 28
±0.5℃；Middle and later periods (39-170h) shaking table temperature of fermenting control is at 32 ± 0.5 DEG C, and humid control is in 40-50%；Incubation time
For 170h；
S3, control of additive raw material: in the fermentation process of step S2, total sugar content is detected when 3d, 4d, 5d, works as total sugar content
Feed supplement is carried out lower than 5%, maintains the control of fermentation liquid total sugar content in 5-7%, until fermentation ends, isolate and purify out from fermentation liquid and tie up
Raw element B12.
In the present embodiment, the preparation steps of female bottle culture medium include: to weigh beet molasses respectively in the step S1
(50%) 110g, dipotassium hydrogen phosphate 0.60g, ammonium sulfate 0.81g, magnesium sulfate 1.65g, manganese sulfate 0.15g, zinc sulfate 0.02g, chlorine
Change cobalt 0.02g, precursor 0.012g, dissolve and be settled to 1000mL, adjusts pH, 6.80-7.40 before disappearing with 20% NaOH solution,
Each 300mL triangular flask dispenses 60mL, and female bottle culture is made after sterilized (0.11-0.12MPa, 121 DEG C, 30min) in wrapping
Base.
In the present embodiment, the preparation steps of Shake flask medium include: to weigh beet molasses respectively suddenly in the step S2
(50%) 170g, sucrose 15g, choline chloride 10.30g, ammonium sulfate 2.60g, magnesium sulfate 1.65g, zinc sulfate 0.08g, cobalt chloride
0.14g, precursor 0.01g, glycerol 1.55g, phosphoglycerol 7g, dissolve and are settled to 1000mL, with 20% NaOH solution tune pH,
6.80-7.40 before disappearing, each 300mL triangular flask dispense 37mL, wrapping, after sterilized (0.11-0.12MPa, 121 DEG C, 30min)
Shake flask medium is made.
In the present embodiment, the preparation steps of supplemented medium include: to weigh sucrose 300g respectively in the step S3,
DMBI 0.20g, CoCl2·6H2O 0.23g dissolves and is settled to 1000mL, adjusts pH to 6.80-7.40, sterilized (0.11-
0.12MPa, 121 DEG C, 30min) after supplemented medium is made.
Through detecting, fermentation ends, vitamin B12 fermentation unit is 199mg/L.
A kind of method for improving denitrified pseudomonas and producing vitamin B12 of comparative example 1
Comparative example 1 is substantially the same manner as Example 3, and difference is: S2, the control of shaking flask incubation: by standard compliant denitrogenation
Pseudomonas alba mother's bottle culture medium aseptically accesses Shake flask medium, inoculum concentration 10%, the control of shaking table temperature 32 ±
0.5℃；Humid control is in 40-50%；Incubation time is about 170h.
Compared with Example 3, shaking flask incubation temperature does not control at 32 ± 0.5 DEG C in fermentation process early period comparative example 1
Temperature is in a relatively low level, and through detecting, fermentation ends, vitamin B12 fermentation unit is 162mg/L.
A kind of method for improving denitrified pseudomonas and producing vitamin B12 of comparative example 2
Comparative example 2 is substantially the same manner as Example 3, and difference is S2, the control of shaking flask incubation: standard compliant denitrogenation is false
(no miscellaneous bacteria, OD value are 0.207) aseptically to access Shake flask medium to pseudomonas bacillus mother's bottle culture medium, and inoculum concentration is
10%, earlier fermentation (0-39h) shaking table temperature is controlled at 25 ± 0.5 DEG C；Middle and later periods (39-170h) shaking table temperature of fermenting control exists
32 ± 0.5 DEG C, humid control is in 40-50%；Incubation time is 170h.
Compared with Example 3, earlier fermentation (0-39h) shaking table temperature is controlled at 25 ± 0.5 DEG C comparative example 1, is detected, is sent out
Ferment terminates, and vitamin B12 fermentation unit is 153mg/L.
The embodiment of the present invention is described above, above description is exemplary, and non-exclusive, and also not
It is limited to disclosed embodiment.Without departing from the scope and spirit of illustrated embodiment, for the art
Many modifications and changes are obvious for those of ordinary skill.
Claims (5)
1. a kind of method for improving denitrified pseudomonas and producing vitamin B12, characterized by the following steps:
S1, female bottle incubation control: the denitrified pseudomonas two generations inclined-plane of activation is scraped into appropriate thallus and is inoculated in female bottle
In culture medium, temperature control is at 27-29 DEG C, and for humid control 30-60%, incubation time was 18-hours for 24 hours；
S2, the control of shaking flask incubation: standard compliant denitrified pseudomonas mother bottle culture medium is aseptically accessed
Shake flask medium, inoculum concentration 10%, earlier fermentation (0-39h) shaking table temperature are controlled at 26-30 DEG C；Ferment middle and later periods (39-
170h) control of shaking table temperature is at 30-34 DEG C, and humid control is 30-60%；Incubation time is 170h；
S3, control of additive raw material: in the fermentation process of step S2, total sugar content is detected when 3d, 4d, 5d, works as total sugar content
Feed supplement is carried out lower than 5%, maintains the control of fermentation liquid total sugar content in 5-7%, until fermentation ends, isolate and purify out from fermentation liquid and tie up
Raw element B12.
2. a kind of method for improving denitrified pseudomonas and producing vitamin B12 as described in claim 1, it is characterised in that: institute
The preparation steps for stating female bottle culture medium in step S1 include: to weigh beet molasses (50%) 110g, dipotassium hydrogen phosphate respectively
0.60g, ammonium sulfate 0.81g, magnesium sulfate 1.65g, manganese sulfate 0.15g, zinc sulfate 0.02g, cobalt chloride 0.02g, precursor 0.012g,
1000mL is dissolved and be settled to, adjusts pH, 6.80-7.40 before disappearing, each 300mL triangular flask packing with 20% NaOH solution
60mL, wrapping, is made female bottle culture medium after sterilized (0.11-0.12MPa, 121 DEG C, 30min).
3. a kind of method for improving denitrified pseudomonas and producing vitamin B12 as described in claim 1, it is characterised in that: institute
The preparation steps for stating Shake flask medium in step S2 include: to weigh beet molasses (50%) 170g, sucrose 15g, chlorination respectively suddenly
Choline 10.30g, ammonium sulfate 2.60g, magnesium sulfate 1.65g, zinc sulfate 0.08g, cobalt chloride 0.14g, precursor 0.01g, glycerol
1.55g, phosphoglycerol 7g, dissolve and are settled to 1000mL, with 20% NaOH solution tune pH, 6.80-7.40 before disappearing, each
300mL triangular flask dispenses 37mL, and Shake flask medium is made after sterilized (0.11-0.12MPa, 121 DEG C, 30min) in wrapping.
4. a kind of method for improving denitrified pseudomonas and producing vitamin B12 as described in claim 1, it is characterised in that: institute
The preparation steps for stating supplemented medium in step S3 include: to weigh sucrose 300g respectively, DMBI 0.20g, CoCl2·6H2O
0.23g dissolves and is settled to 1000mL, pH to 6.80-7.40 is adjusted, after sterilized (0.11-0.12MPa, 121 DEG C, 30min)
Supplemented medium is made.
5. a kind of method for improving denitrified pseudomonas and producing vitamin B12 as described in claim 1, it is characterised in that: institute
State the feature of standard compliant denitrified pseudomonas mother bottle culture medium in step S2 are as follows: without miscellaneous bacteria, OD value is 0.207-
0.220。
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| CN111073922A (en) * | 2019-12-26 | 2020-04-28 | 湖北广济药业股份有限公司 | Vitamin B12Fermentation supplementing medium and supplementing method |
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