CN107164420A - A kind of method of L alanine semicontinuous fermentation - Google Patents
A kind of method of L alanine semicontinuous fermentation Download PDFInfo
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- CN107164420A CN107164420A CN201710552493.3A CN201710552493A CN107164420A CN 107164420 A CN107164420 A CN 107164420A CN 201710552493 A CN201710552493 A CN 201710552493A CN 107164420 A CN107164420 A CN 107164420A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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Abstract
The present invention relates to fermentation engineering field, a kind of method of L alanine semicontinuous fermentation is specifically disclosed.Methods described, comprises the following steps：It is prepared by seed liquor；It is prepared by culture medium；Semicontinuous fermentation culture L alanine；Zymotic fluid is collected, purification obtains L alanine.The method of L alanine semicontinuous fermentation provided by the present invention, technique is simple, environmental protection, eliminates to the step of the handling that carried out disinfection per a batch of seed liquor, fermentation tank and culture medium, time saving and energy saving, energy consumption is low, and yield is high.
Description
Technical field
The invention belongs to fermentation engineering field, more particularly to a kind of method of ALANINE semicontinuous fermentation.
Background technology
ALANINE is colourless to white crystalline powder, water, ethanol is dissolved in, insoluble in ether and acetone.It is mainly used in life
Change research, tissue cultures, liver functional testing, flavoring agent, the flavor effect that flavouring can be increased, also act as tart flavour corrigent,
Improve the tart flavour of organic acid.
However, the fermentation method of existing ALANINE, many methods using batch fermentation are, it is necessary to respectively to per a batch of
Seed liquor, fermentation tank and culture medium carry out disinfection processing, cause the waste of energy consumption, yield poorly.
The content of the invention
Wasted for the energy consumption that prior art is present, the problems such as yielding poorly, it is semicontinuous that the present invention provides a kind of ALANINE
The method of fermentation.
In order to solve the above technical problems, the technical solution used in the present invention is：
A kind of method of ALANINE semicontinuous fermentation, at least comprises the following steps：
（1）Shake-flask seed is inoculated into seeding tank, aseptic compressed air is passed through, 35 ~ 38 DEG C, cultivates 4 ~ 7h, obtain seed liquor；
（2）120 ~ 180g/L glucose and component A are provided, the glucose is separated into progress at 120 ~ 122 DEG C with the component A
Disinfect, be cooled to after less than 60 DEG C, the glucose is added to component A, culture medium is obtained, wherein, the component A bags
Include：5 ~ 10g/L yeast extraction powder, 5 ~ 10g/L peptones, 0.25 ~ 0.5g/L potassium dihydrogen phosphates, 0.2 ~ 0.6g/L magnesium sulfate, 1 ~
2g/L ammonium phosphate, 0.05 ~ 0.1g/L defoamers；
（3）120 ~ 122 DEG C are carried out with saturated steam to fermentation tank to disinfect, the culture medium and seed liquor added after sterilization,
The volume ratio of the culture medium and seed liquor is 20:1-4, with ammoniacal liquor or sulphur acid for adjusting pH to 7.0 ± 0.2, is passed through sterile compression
Air, 35 ~ 38 DEG C, 4 ~ 7h of stir culture stops ventilation, continues after 16 ~ 20h of stirring reaction, and reaction system body is discharged every 4h
The zymotic fluid of product 25%, while adding the fresh culture of initial reaction system volume 20%；Initial reaction system is accessed every 8h
The seed liquor of volume 10%, reacts 9 days；
（4）Zymotic fluid is collected, purification obtains ALANINE.
The method of the ALANINE semicontinuous fermentation provided relative to prior art, the present invention, technique is simple, green ring
Protect, the step of fermentation tank for eliminating 90% batch is disinfected, time saving and energy saving, energy consumption is low, yield is high.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The embodiment of the present invention provides a kind of method of ALANINE semicontinuous fermentation, at least comprises the following steps：
（1）Shake-flask seed is inoculated into seeding tank, aseptic compressed air is passed through, 35 ~ 38 DEG C, cultivates 4 ~ 7h, obtain seed liquor；
（2）120 ~ 180g/L glucose and component A are provided, the glucose is separated into progress at 120 ~ 122 DEG C with the component A
Disinfect, be cooled to after less than 60 DEG C, the glucose is added to component A, culture medium is obtained, wherein, the component A bags
Include：5 ~ 10g/L yeast extraction powder, 5 ~ 10g/L peptones, 0.25 ~ 0.5g/L potassium dihydrogen phosphates, 0.2 ~ 0.6g/L magnesium sulfate, 1 ~
2g/L ammonium phosphate, 0.05 ~ 0.1g/L defoamers；
（3）120 ~ 122 DEG C are carried out with saturated steam to fermentation tank to disinfect, the culture medium and seed liquor added after sterilization,
The volume ratio of the culture medium and seed liquor is 20:1-4, with ammoniacal liquor or sulphur acid for adjusting pH to 7.0 ± 0.2, is passed through sterile compression
Air, 35 ~ 38 DEG C, 4 ~ 7h of stir culture stops ventilation, continues after 16 ~ 20h of stirring reaction, and reaction system body is discharged every 4h
The zymotic fluid of product 25%, while adding the fresh culture of initial reaction system volume 20%；Initial reaction system is accessed every 8h
The seed liquor of volume 10%, reacts 9 days；
（4）Zymotic fluid is collected, purification obtains ALANINE.
Specifically, aseptic compressed air is passed through, while dissolved oxygen is provided, gnotobasis is maintained；35 ~ 38 DEG C, culture 4 ~
7h, makes OD600 values reach 30 ~ 40, inoculum concentration reaches 10%, obtains required seed liquor.
Specifically, by glucose and component A, carry out disinfection processing at 120 ~ 122 DEG C respectively, is cooled to less than 60 DEG C, will
Glucose is added to component A, is that, in order to prevent more than 60 DEG C, glucose chemically reacts with the component in component A.
Specifically, aseptic compressed air is passed through, while dissolved oxygen is provided, gnotobasis is maintained；4 ~ 7h of stir culture, makes
OD values reach 10 ~ 15；Stop ventilation, be to enter anaerobic stages.
Specifically, all zymotic fluids discharged in 9 days are collected, the purification processes for entering series obtain ALANINE.
Explanation is further explained to above-mentioned preparation method below：
Preferably, step（1）In, it is 1 to be passed through aseptic compressed air amount:0.4 ~ 1.5 vvm, while dissolved oxygen is provided, is maintained
Gnotobasis.
Preferably, glucose and component A are the aqueous solution, environmental protection.
Preferably, defoamer is bubble enemy, with non-toxic efficient, high temperature resistant, suds time long feature, can meet whole fermentation
The requirement of process froth breaking.
Preferably, the time is disinfected for 20 ~ 25min, it is ensured that while sterilization is thorough, reduce energy consumption.
Preferably, step（3）In, the culture medium after sterilization is 3.6t, and seed liquor is 180-720L；It is passed through sterile compression empty
Tolerance is 1:0.5 ~ 1.0vvm, while dissolved oxygen is provided, maintains gnotobasis.
Preferably, the mixing speed of stir culture is 150 ~ 200rpm, is rapidly achieved the dissolved oxygen amount needed for culture.
Preferably, the mixing speed of stirring reaction is 50 ~ 100rpm, reaction system is stirred.
This method technique is simple, and environmental protection, the step of fermentation tank for eliminating 90% batch is disinfected is time saving and energy saving,
Energy consumption is low, and yield is high.
Method in order to better illustrate ALANINE semicontinuous fermentation provided in an embodiment of the present invention, below by implementation
Example is further to be illustrated.
Embodiment 1
A kind of method of ALANINE semicontinuous fermentation, comprises the following steps：
（1）Shake-flask seed is inoculated into 800L seeding tanks, aseptic compressed air 1 is passed through：1vvm, cultivates 6h, OD600 values by 37 DEG C
For 35, seed liquor is obtained；
（2）By 120g/L glucose and component A：10g/L yeast extraction powder, 8g/L peptones, 0.25g/L potassium dihydrogen phosphates,
0.6g/L magnesium sulfate, 2g/L ammonium phosphate, 0.05g/L bubble enemies, carry out disinfection processing at 120 ~ 122 DEG C respectively, be cooled to 60 DEG C with
Under, glucose is added to component A, culture medium is obtained：
（3）120-122 DEG C is carried out to 5t fermentation tanks with saturated steam and disinfects 20min, the culture medium added after sterilization
3.6t, seed liquor 400L, adjust pH to 6.8 with ammoniacal liquor, are passed through aseptic compressed air 1：1.0vvm, is stirred with 150rpm by 35 DEG C
7h is cultivated, OD values reach 12, stop ventilation, continue with 50rpm after stirring reaction 20h, every 4h discharge reaction systems volume 25%
Zymotic fluid, while adding the fresh culture of initial reaction system volume 20%；Initial reaction system volume is accessed every 8h
10% seed liquor, reacts 9 days；
（4）Zymotic fluid is collected, purification obtains ALANINE.
Embodiment 2
A kind of method of ALANINE semicontinuous fermentation, comprises the following steps：
（1）Shake-flask seed is inoculated into 800L seeding tanks, aseptic compressed air 1 is passed through：1.5vvm, cultivates 7h, OD600 by 38 DEG C
It is worth for 40, obtains seed liquor；
（2）By 180g/L glucose and component A：8g/L yeast extraction powder, 5g/L peptones, 0.5g/L potassium dihydrogen phosphates, 0.5g/
L magnesium sulfate, 1.5g/L ammonium phosphate, 0.1g/L bubble enemies, carry out disinfection processing at 120 ~ 122 DEG C respectively, is cooled to less than 60 DEG C, will
Glucose is added to component A, obtains culture medium：
（3）120-122 DEG C is carried out to 5t fermentation tanks with saturated steam and disinfects 23min, the culture medium added after sterilization
3.6t, seed liquor 180L, adjust pH to 7 with ammoniacal liquor, are passed through aseptic compressed air 1：0.7vvm, is stirred with 200rpm and trained by 36 DEG C
4h is supported, OD values reach 10, stop ventilation, continue to stir anti-with 100rpm:After 16h, reaction system volume 25% is discharged every 4h
Zymotic fluid, while adding the fresh culture of initial reaction system volume 20%；Every 8h access initial reaction systems volume 10%
Seed liquor, react 9 days；
（4）Zymotic fluid is collected, purification obtains ALANINE.
Embodiment 3
A kind of method of ALANINE semicontinuous fermentation, comprises the following steps：
（1）Shake-flask seed is inoculated into 800L seeding tanks, aseptic compressed air 1 is passed through：0.4vvm, cultivates 4h, OD600 by 35 DEG C
It is worth for 30, obtains seed liquor；
（2）By 150g/L glucose and component A：5g/L yeast extraction powder, 10g/L peptones, 0.3g/L potassium dihydrogen phosphates,
0.2g/L magnesium sulfate, 2g/L ammonium phosphate, 0.06g/L bubble enemies, carry out disinfection processing at 120 ~ 122 DEG C respectively, be cooled to 60 DEG C with
Under, glucose is added to component A, culture medium is obtained：
（3）120-122 DEG C is carried out to 5t fermentation tanks with saturated steam and disinfects 25min, the culture medium added after sterilization
3.6t, seed liquor 720L, with sulphur acid for adjusting pH to 7.2, are passed through aseptic compressed air 1：0.5vvm, is stirred with 180rpm by 38 DEG C
5h is cultivated, OD values reach 15, stop ventilation, continue with 80rpm after stirring reaction 18h, every 4h discharge reaction systems volume 25%
Zymotic fluid, while adding the fresh culture of initial reaction system volume 20%；Initial reaction system volume is accessed every 8h
10% seed liquor, reacts 9 days；
（4）Zymotic fluid is collected, purification obtains ALANINE.
For the characteristic of the method that better illustrates a kind of ALANINE semicontinuous fermentation provided in an embodiment of the present invention, under
The yield for the ALANINE that face prepares embodiment 1,2,3 with fermenting in batches（Comparative example）The yield of gained ALANINE is carried out
Contrast, as a result as shown in table 1.
Table 1
| Contrast project | Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example |
| Yield（Ton） | 4 | 3.8 | 3.9 | 1.5 |
In terms of 10 batch batch fermentation yield, embodiment 1,2,3 is prepared into quantity of steam that ALANINE consumed with sending out in batches
Ferment（Comparative example）The quantity of steam consumed is contrasted, as a result as shown in table 2.
Table 2
| Contrast project | Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example |
| Consume steam（Ton） | 20 | 22 | 21 | 32 |
It can be obtained by data above, the method for ALANINE semicontinuous fermentation provided by the present invention, with energy consumption is saved, yield is high
Advantage, save 10-12 tons of energy consumption, 2.3-2.5 tons of output increased.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
Any modification, equivalent substitution or improvement made within refreshing and principle etc., should be included in the scope of the protection.
Claims (8)
1. a kind of method of ALANINE semicontinuous fermentation, it is characterised in that：At least comprise the following steps：
（1）Shake-flask seed is inoculated into seeding tank, aseptic compressed air is passed through, 35 ~ 38 DEG C, cultivates 4 ~ 7h, obtain seed liquor；
（2）120 ~ 180g/L glucose and component A are provided, the glucose is separated into progress at 120 ~ 122 DEG C with the component A
Disinfect, be cooled to after less than 60 DEG C, the glucose is added to component A, culture medium is obtained, wherein, the component A bags
Include：5 ~ 10g/L yeast extraction powder, 5 ~ 10g/L peptones, 0.25 ~ 0.5g/L potassium dihydrogen phosphates, 0.2 ~ 0.6g/L magnesium sulfate, 1 ~
2g/L ammonium phosphate, 0.05 ~ 0.1g/L defoamers；
（3）120 ~ 122 DEG C are carried out with saturated steam to fermentation tank to disinfect, the culture medium and seed liquor added after sterilization,
The volume ratio of the culture medium and seed liquor is 20:1-4, with ammoniacal liquor or sulphur acid for adjusting pH to 7.0 ± 0.2, is passed through sterile compression
Air, 35 ~ 38 DEG C, 4 ~ 7h of stir culture stops ventilation, continues after 16 ~ 20h of stirring reaction, and reaction system body is discharged every 4h
The zymotic fluid of product 25%, while adding the fresh culture of initial reaction system volume 20%；Initial reaction system is accessed every 8h
The seed liquor of volume 10%, reacts 9 days；
（4）Zymotic fluid is collected, purification obtains ALANINE.
2. the method for ALANINE semicontinuous fermentation as claimed in claim 1, it is characterised in that：The step（1）In, it is passed through
Aseptic compressed air amount is 1:0.4~1.5 vvm.
3. the method for ALANINE semicontinuous fermentation as claimed in claim 1, it is characterised in that：The glucose and component A
It is the aqueous solution.
4. the method for ALANINE semicontinuous fermentation as claimed in claim 1, it is characterised in that：The defoamer is bubble enemy.
5. the method for ALANINE semicontinuous fermentation as claimed in claim 1, it is characterised in that：The time of disinfecting is
20~25min。
6. the method for ALANINE semicontinuous fermentation as claimed in claim 1, it is characterised in that：The step（3）In, sterilization
Culture medium afterwards is 3.6t, and seed liquor is 180-720L；It is 1 to be passed through aseptic compressed air amount:0.5~1.0vvm.
7. the method for ALANINE semicontinuous fermentation as claimed in claim 1, it is characterised in that：The stirring of the stir culture
Speed is 150 ~ 200rpm.
8. the method for ALANINE semicontinuous fermentation as claimed in claim 1, it is characterised in that：The stirring of the stirring reaction
Speed is 50 ~ 100rpm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438184A (en) * | 2019-08-26 | 2019-11-12 | 山东润德生物科技有限公司 | A kind of method of N-acetylglucosamine content and conversion ratio in raising fermentation liquid |
| CN111574390A (en) * | 2020-05-21 | 2020-08-25 | 赵兰坤 | Efficient green production and extraction process of amino acid |
| CN114015559A (en) * | 2021-09-08 | 2022-02-08 | 安徽华恒生物科技股份有限公司 | High-efficiency valine semi-continuous fermentation method and complete equipment thereof |
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| CN106047740A (en) * | 2016-01-14 | 2016-10-26 | 齐鲁工业大学 | Bacterial strain capable of realizing high yield of L-alanine |
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| WO2010115838A1 (en) * | 2009-04-03 | 2010-10-14 | Dsm Ip Assets B.V. | Fermentation process |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438184A (en) * | 2019-08-26 | 2019-11-12 | 山东润德生物科技有限公司 | A kind of method of N-acetylglucosamine content and conversion ratio in raising fermentation liquid |
| CN111574390A (en) * | 2020-05-21 | 2020-08-25 | 赵兰坤 | Efficient green production and extraction process of amino acid |
| CN114015559A (en) * | 2021-09-08 | 2022-02-08 | 安徽华恒生物科技股份有限公司 | High-efficiency valine semi-continuous fermentation method and complete equipment thereof |
| CN114015559B (en) * | 2021-09-08 | 2023-06-23 | 安徽华恒生物科技股份有限公司 | Efficient valine semi-continuous fermentation method and complete equipment thereof |
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Application publication date: 20170915 |