CN108796025A - A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique - Google Patents
A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique Download PDFInfo
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- CN108796025A CN108796025A CN201810597193.1A CN201810597193A CN108796025A CN 108796025 A CN108796025 A CN 108796025A CN 201810597193 A CN201810597193 A CN 201810597193A CN 108796025 A CN108796025 A CN 108796025A
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- zymotic fluid
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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
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
- C12P7/60—2-Ketogulonic acid
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Abstract
A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique, the fermentation of first step single bacterium：D-glucitol is converted to L- sorboses by a step strain；Second step mixed fermentation：Start that only the product sorbose mash that the part first step is fermented directly is incorporated into two step culture mediums, make the initial sorbose concentration only up to 2.0~9.0% of two step culture mediums, then remaining sorbose mash is added during fermentation again, zymotic fluid maintains suitable osmotic pressure during making fermentation, it is aided with the regulation and control of the segmentation to zymotic fluid pH value again, achieve the effect that inhibit a step growth to breed in second step fermentation process and promote two step culture propagations and metabolism, the technique eliminates the process that low temperature sterilization post-processing is carried out to first step tunning, reduce destruction of the high temperature to sorbose ingredient, the mixed fermentation period can be shortened, simultaneously, cancel the sterilization treatment to the lye used in second step fermentation process, the consumption of manpower and power in sterilization process can be reduced, section drop production cost.
Description
Technical field
The present invention relates to a kind of improved 2-keto-L-gulonic acid fermentation manufacturing techniques, belong to microbial fermentation technology neck
Domain.
Background technology
Vitamin C (Vitamin C, abbreviation Vc) is also known as L-AA (L-Ascorbic acid), in medicine and food
All occupy critically important status in industry, " two-step fermenting " for generally using China's independent research domestic at present produces, " two steps
The first step of fermentation method " is single bacterium fermentation：1. D-glucitol is converted to L- sorboses by a step strain, 2. to sorbose wine with dregs
Liquid carries out 70~80 DEG C of low temperature sterilization post-processing in 30 minutes, kills the step strain in sorbose mash；Second step is mixing
Bacterium is fermented：L- sorboses are converted to 2-keto-L-gulonic acid (2-KGA) by two step strains；2-keto-L-gulonic acid passes through again
It crosses and extracts and be converted into vitamin C.
During " two-step fermenting " production is ascorbic, the product L- sorboses of first step fermentation are as second step
The substrate of fermentation will first carry out at 30 minutes 70~80 DEG C of low temperature sterilizations sorbose mash after fermentation in the first step
Then it disposably could all be incorporated into two step culture mediums by reason, and closed in the product sorbose mash that the first step is fermented
After entering into two step culture mediums, the initial sorbose concentration of two step culture mediums is up to 11~14%；And in second step fermentation
In the process in order to maintain certain acid-base balance, the metabolism of strain is promoted to produce acid, sterilized treated sterile carbonic acid need to be used
Sodium solution adjusts pH value.Such production of vitamin C technique：1, the low temperature that the first step after fermentation carries out sorbose mash
Sterilization treatment destroys the nutritional ingredient of sorbose；2, the product sorbose mash of first step fermentation is disposably whole
It is incorporated into two step culture mediums, causes the Initial sugar concentration of two step culture mediums higher, because strain is to the adaptability of high glucose concentration
It is limited with tolerance, cause the lag phase of growth long, extend fermentation period, and limit terminal acid into one
Step improves；3, adjust the pH value of two-step fermentation liquid using sterile sodium carbonate solution, the preparation of sodium carbonate liquor, sterilizing in manpower and
Consumption is more in terms of power.
It, need to be to the fermentation manufacturing technique of 2-keto-L-gulonic acid in order to save cost declining, raising production efficiency, enhance production capacities
It is improved.
Invention content
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, and provide " two-step fermenting " production of vitamin C
A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique of technology drops 2-keto-L-gulonic acid fermenting and producing cost with section,
And its production efficiency is improved, promote the progress of " two-step fermenting " Progresses of Vitamin C Productive Technology.
The present invention solves the technical solution that its technical problem is taken：A kind of improved 2-keto-L-gulonic acid fermentation
Production technology, it is as follows：
The first step, single bacterium fermentation
D-glucitol is converted to L- sorboses by a step strain；
Second step, mixed fermentation
The product sorbose mash that the part first step is fermented directly is incorporated into two step culture mediums, until two step culture mediums
Initial sorbose concentration is inoculated with two step strains, culture, when the residual sorbose concentration in zymotic fluid is down to up to 2.0~9.0%
When 1.0~5.0%, proceeds by the continuous of remaining sorbose mash and add, and in fermentation process thereafter, it is surplus by adding
Remaining sorbose mash maintains the residual sorbose concentration of zymotic fluid 0.5~4.0%, and fermentation stops adding before terminating, until fermentation
Fermentation is terminated when residual sorbose concentration≤0.5mg/mL of liquid, and temperature is controlled during entire mixed fermentation 28~33
DEG C, adjust the pH value of zymotic fluid using 15~25% sodium hydroxide solution：
(1), it is 7.5~8.5 that the starting stage, which maintains the pH value of zymotic fluid,；
(2), when the content of 2-keto-L-gulonic acid in zymotic fluid is up to 5~15mg/ml, the pH value of zymotic fluid is lowered
To 6.5~7.0；
(3), after the content of 2-keto-L-gulonic acid in zymotic fluid is up to 50~90mg/ml, the pH value of zymotic fluid is adjusted
It is 7.0~7.5, to terminal, produces 2-keto-L-gulonic acid.
This improved 2-keto-L-gulonic acid fermentation manufacturing technique provided by the present invention, 1, eliminate to the first step
Tunning sorbose mash carries out the process of low temperature sterilization post-processing, and hair is turned up in the starting stage by fermenting in second step
The means of zymotic fluid pH value inhibit the growth and breeding of a step strain in zymotic fluid, to reach quickly drop one step lower bacterial activity
Purpose；2, when the residual sorbose concentration in two-step fermentation liquid is down to 1.0~5.0%, remaining sorbose mash is proceeded by
It is continuous add, and in fermentation process thereafter, the residual sorbose of zymotic fluid is maintained by adding remaining sorbose mash
Concentration is 0.5~4.0%, when the content of 2-keto-L-gulonic acid in zymotic fluid is up to 5~15mg/ml, by the pH value of zymotic fluid
6.5~7.0 are adjusted downward to, so that zymotic fluid is released the inhibiting effect that high glucose concentration grows microorganism, only maintains suitable osmotic pressure,
And give two step growth and breed optimum pH environment, promote the growth and breeding of two step strains；3, in second step fermentation
Final stage adjusts the pH value of zymotic fluid to 7.0~7.5, promotes the metabolism production acid of two step strains；4, in entire mixed fermentation
During, substitute the pH that sodium carbonate liquor in the prior art adjusts zymotic fluid using 15~25% sodium hydroxide solution
Value, since Sodium Hydroxide Alkaline is strong, almost without microbial survival in 15~25% solution, because without the hydrogen-oxygen to using
Change sodium solution and carries out sterilization treatment.
What the present invention obtained has the beneficial effect that：
1, cancel the process for carrying out low temperature sterilization post-processing to first step tunning sorbose mash, reduce high temperature pair
The destruction of sorbose ingredient can shorten the mixed fermentation period, meanwhile, cancel to the lye that is used in second step fermentation process
Sterilization treatment can reduce the consumption of manpower and power in sterilization process, section drop production cost.
2, start that only the product sorbose mash that the part first step is fermented is incorporated into two step culture mediums, make two step cultures
The initial sorbose concentration only up to 2.0~9.0% of base, remaining sorbose mash is then added during fermentation, is made again
Zymotic fluid maintains suitable osmotic pressure during fermentation, then is aided with the regulation and control of the segmentation to zymotic fluid pH value, reaches in second step
Inhibit a step growth to breed in fermentation process and the effect of two step culture propagations and metabolism of promotion, fermentation termination 2- ketone can be made
Base-L- Colognes acid content is promoted to 130.0~155.0mg/ml by 93.0~103.0mg/ml of prior art, rate of producing acid by
2.1~2.8g/Lh is promoted to 3.0~3.7g/Lh, realizes the efficient production of 2-keto-L-gulonic acid, is conducive to improve equipment
Utilization rate increases production capacity.
Specific implementation mode
Following instance is for illustrating the present invention, but protection scope of the present invention is not limited only to this, it is required that
The range of protection is recorded in the claim of claim.
Embodiment 1：
A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique, it is as follows：
The first step, single bacterium fermentation
D-glucitol is converted to L- sorboses by a step strain；
Second step, mixed fermentation
Two step culture medium prescriptions (%)：Corn steep liquor 1.5, urea 0.1, potassium dihydrogen phosphate 0.05, magnesium sulfate 0.02, pH6.5
~8.2；
The product sorbose mash directly (being handled without low temperature sterilization) that the part first step is fermented is incorporated into two step cultures
In base, until the initial sorbose concentration of two step culture mediums is inoculated with two step strains, culture, when the residual mountain in zymotic fluid up to 9.0%
When pears sugar concentration is down to 3.5~5.0%, proceeds by the continuous of the remaining sorbose mash handled without low temperature sterilization and adds,
And in fermentation process thereafter, the residual of zymotic fluid is maintained by adding the remaining sorbose mash handled without low temperature sterilization
2.0~4.0%, fermentation terminates to be stopped adding sorbose concentration for first 7~15 hours, until the residual sorbose concentration of zymotic fluid≤
Fermentation is terminated when 0.5mg/mL, and temperature is controlled during entire mixed fermentation at 28~33 DEG C, uses 15~25% hydrogen-oxygen
Change the pH value that sodium solution adjusts zymotic fluid：
(1), it is 7.6~8.2 that the starting stage, which maintains the pH value of zymotic fluid,；
(2), when the content of 2-keto-L-gulonic acid in zymotic fluid is up to 5~15mg/ml, the pH value of zymotic fluid is lowered
To 6.5~7.0；
(3), after the content of 2-keto-L-gulonic acid in zymotic fluid is up to 50~90mg/ml, the pH value of zymotic fluid is adjusted
It is 7.0~7.5, to terminal, produces 2-keto-L-gulonic acid.
Mixed fermentation result：Fermentation period 45h, a concentration of 139.57mg/ml of 2-keto-L-gulonic acid, rate of producing acid
3.10g/Lh, sterility are negative.
Embodiment 2：
A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique, it is as follows：
The first step, single bacterium fermentation
D-glucitol is converted to L- sorboses by a step strain；
Second step, mixed fermentation
Two step culture medium prescriptions (%)：Corn steep liquor 1.5, urea 0.1, potassium dihydrogen phosphate 0.05, magnesium sulfate 0.02, pH6.5
~8.2；
The product sorbose mash directly (being handled without low temperature sterilization) that the part first step is fermented is incorporated into two step cultures
In base, until the initial sorbose concentration of two step culture mediums is inoculated with two step strains, culture, when the residual mountain in zymotic fluid up to 2.5%
When pears sugar concentration is down to 1.0~2.0%, proceeds by the continuous of the remaining sorbose mash handled without low temperature sterilization and adds,
And in fermentation process thereafter, the residual of zymotic fluid is maintained by adding the remaining sorbose mash handled without low temperature sterilization
0.5~2.0%, fermentation terminates to be stopped adding sorbose concentration for first 3~8 hours, until the residual sorbose concentration of zymotic fluid≤
Fermentation is terminated when 0.5mg/mL, and temperature is controlled during entire mixed fermentation at 28~33 DEG C, uses 15~25% hydrogen-oxygen
Change the pH value that sodium solution adjusts zymotic fluid：
(1), it is 7.7~8.2 that the starting stage, which maintains the pH value of zymotic fluid,；
(2), when the content of 2-keto-L-gulonic acid in zymotic fluid is up to 5~15mg/ml, the pH value of zymotic fluid is lowered
To 6.5~7.0；
(3), after the content of 2-keto-L-gulonic acid in zymotic fluid is up to 50~90mg/ml, the pH value of zymotic fluid is adjusted
It is 7.0~7.5, to terminal, produces 2-keto-L-gulonic acid.
Mixed fermentation result：Fermentation period 43h, a concentration of 146.88mg/ml of 2-keto-L-gulonic acid, rate of producing acid
3.42g/Lh, sterility are negative.
Above example is the preferred embodiment of the present invention, to the 2-keto-L-gulonic acid production strain of different Pseudomonas
Corresponding adjustment must be made under the premise of above example is conceived, these adjustment also should be regarded as protection scope of the present invention.
Claims (4)
1. a kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique, it is characterised in that it is as follows：
The first step, single bacterium fermentation
D-glucitol is converted to L- sorboses by a step strain；
Second step, mixed fermentation
The product sorbose mash that the part first step is fermented directly is incorporated into two step culture mediums, until two step culture mediums is initial
Sorbose concentration is inoculated with two step strains up to 2.0~9.0%, culture, when the residual sorbose concentration in zymotic fluid is down to 1.0~
When 5.0%, proceeds by the continuous of remaining sorbose mash and add, and in fermentation process thereafter, by adding remaining mountain
Pears sugar mash maintains the residual sorbose concentration of zymotic fluid 0.5~4.0%, and fermentation stops adding before terminating, until zymotic fluid
Fermentation is terminated when remaining sorbose concentration≤0.5mg/mL, temperature is controlled during entire mixed fermentation at 28~33 DEG C, is made
The pH value of zymotic fluid is adjusted with 15~25% sodium hydroxide solution：
(1), it is 7.5~8.5 that the starting stage, which maintains the pH value of zymotic fluid,；
(2), when the content of 2-keto-L-gulonic acid in zymotic fluid is up to 5~15mg/ml, the pH value of zymotic fluid is adjusted downward to 6.5
~7.0；
(3), after the content of 2-keto-L-gulonic acid in zymotic fluid is up to 50~90mg/ml, the pH value of zymotic fluid is adjusted to
7.0~7.5, to terminal, produce 2-keto-L-gulonic acid.
2. improved 2-keto-L-gulonic acid fermentation manufacturing technique according to claim 1, it is characterised in that：
Second step, mixed fermentation
Two step culture medium prescriptions (%)：Corn steep liquor 1.5, urea 0.1, potassium dihydrogen phosphate 0.05, magnesium sulfate 0.02, pH6.5~
8.2。
3. improved 2-keto-L-gulonic acid fermentation manufacturing technique according to claim 1 or 2, it is characterised in that：
Second step, mixed fermentation
The product sorbose mash that the part first step is fermented directly is incorporated into two step culture mediums, until two step culture mediums is initial
Sorbose concentration is inoculated with two step strains, culture, when the residual sorbose concentration in zymotic fluid is down to 3.5~5.0% up to 9.0%
When, it proceeds by the continuous of remaining sorbose mash and adds, and in fermentation process thereafter, by adding remaining sorbose wine with dregs
Liquid maintains the residual sorbose concentration of zymotic fluid 2.0~4.0%, and fermentation terminates to be stopped adding for first 7~15 hours, until zymotic fluid
Residual sorbose concentration≤0.5mg/mL when terminate fermentation, during entire mixed fermentation control temperature at 28~33 DEG C,
The pH value of zymotic fluid is adjusted using 15~25% sodium hydroxide solution：
(1), it is 7.6~8.2 that the starting stage, which maintains the pH value of zymotic fluid,；
(2), when the content of 2-keto-L-gulonic acid in zymotic fluid is up to 5~15mg/ml, the pH value of zymotic fluid is adjusted downward to 6.5
~7.0；
(3), after the content of 2-keto-L-gulonic acid in zymotic fluid is up to 50~90mg/ml, the pH value of zymotic fluid is adjusted to
7.0~7.5, to terminal, produce 2-keto-L-gulonic acid.
4. improved 2-keto-L-gulonic acid fermentation manufacturing technique according to claim 1 or 2, it is characterised in that：
Second step, mixed fermentation
The product sorbose mash that the part first step is fermented directly is incorporated into two step culture mediums, until two step culture mediums is initial
Sorbose concentration is inoculated with two step strains, culture, when the residual sorbose concentration in zymotic fluid is down to 1.0~2.0% up to 2.5%
When, it proceeds by the continuous of remaining sorbose mash and adds, and in fermentation process thereafter, by adding remaining sorbose wine with dregs
Liquid maintains the residual sorbose concentration of zymotic fluid 0.5~2.0%, and fermentation terminates to be stopped adding for first 3~8 hours, until zymotic fluid
Residual sorbose concentration≤0.5mg/mL when terminate fermentation, during entire mixed fermentation control temperature at 28~33 DEG C,
The pH value of zymotic fluid is adjusted using 15~25% sodium hydroxide solution：
(1), it is 7.7~8.2 that the starting stage, which maintains the pH value of zymotic fluid,；
(2), when the content of 2-keto-L-gulonic acid in zymotic fluid is up to 5~15mg/ml, the pH value of zymotic fluid is adjusted downward to 6.5
~7.0；
(3), after the content of 2-keto-L-gulonic acid in zymotic fluid is up to 50~90mg/ml, the pH value of zymotic fluid is adjusted to
7.0~7.5, to terminal, produce 2-keto-L-gulonic acid.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000514285A (en) * | 1996-10-24 | 2000-10-31 | アーチャー―ダニエルズ―ミッドランド カンパニー | Novel bacterial strains and their use in fermentation processes for 2-keto-L-gulonic acid production |
| CN106434830A (en) * | 2016-12-20 | 2017-02-22 | 宁夏启元药业有限公司 | Method for improving 2-keto-L-gluconic acid fermentation efficiency |
-
2018
- 2018-06-11 CN CN201810597193.1A patent/CN108796025A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000514285A (en) * | 1996-10-24 | 2000-10-31 | アーチャー―ダニエルズ―ミッドランド カンパニー | Novel bacterial strains and their use in fermentation processes for 2-keto-L-gulonic acid production |
| CN106434830A (en) * | 2016-12-20 | 2017-02-22 | 宁夏启元药业有限公司 | Method for improving 2-keto-L-gluconic acid fermentation efficiency |
Non-Patent Citations (3)
| Title |
|---|
| 张静等: "分阶段pH调控提高2-酮基-L-古龙酸生产", 《生物工程学报》 * |
| 王沛等: "《制药工艺学》", 31 August 2017, 中国中医药出版社 * |
| 翟兵兵等: "VC三菌种一步发酵方法的构建与研究", 《中国生物工程杂志》 * |
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Application publication date: 20181113 |