CN107955828A - A kind of Vitamin C Two-step Fermentation production method - Google Patents
A kind of Vitamin C Two-step Fermentation production method Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
The present invention relates to a kind of Vitamin C Two-step Fermentation production method, this method includes the three-level seed culture of the mixed culture to being made of bacillus megaterium and raw ketone group 2-KLG bacterium, feature is to be directly connected to cultured three-level seed liquor to carry out fermented and cultured in the fermentation medium that sterilization treatment crosses, in fermentation, stream plus L sorboses, fermentation medium are matched with urea, biotin, corn pulp, potassium dihydrogen phosphate and magnesium sulfate at the very start at the same time.The feeding strategy that the present invention passes through instant stream plus sorbose after the conjunction sugar operation in fermentation medium after optimizing fermentation medium composition, having deleted sterilizing, culture transferring, so as to improve mixed bacterium growth metabolism environment, rate of producing acid is finally improved, Cologne acid yield is improved, improves and sent out ferment conversion ratio, reduce material residual during fermentation ends, improve zymotic fluid quality, and shorten fermentation period.
Description
Technical field
The present invention relates to biology and new medical technology, more particularly to a kind of Vitamin C Two-step Fermentation production method.
Background technology
Vitamin C also known as L-ascorbic acid, are a kind of water soluble vitamins, have important physiological function, purposes is very
Extensively.Currently, vitamin C yield in China's is huge, accounts for more than the 90% of Gross World Product, wherein yield 80% is with upper outlet.Production
Based on " two-step fermenting ", the first step is under the effect of black vinegar acidfast bacilli, and D-glucitol is oxidized to L- sorbs for the method country
Sugar, is commonly called as alcohol sugar conversion；This step zymotechnique maturation and high conversion rate, can be up to more than 98%；It is related to it both at home and abroad at present
Gene and conversion base Quality Research are clearer；Second step is by bacillus megaterium (being commonly called as big bacterium) and ordinary student ketone group
L~sorbose is further oxidized to 2- keto-L-gulonics under the action of the mixed culture of 2-KLG bacterium (being commonly called as small bacterium) composition
Acid, is commonly called as saccharic acid conversion.Concomitance bacterium is grown in advance during mixed fermentation, and metabolism discharges biologically active albumen, makees therewith
For acid-producing bacteria, start its growth, production acid.I.e. the growth of acid-producing bacteria and acid producing ability are largely by concomitance bacterium growth metabolism
Restriction, therefore by improving formula, optimizing big bacterium growing environment, adjust big bacterium physiological status, promote mixed bacterium metabolism, for carrying
High 2-keto-L-gulonic acid fermentation index, raising production capacity, reduction production cost have conclusive effect.
The content of the invention
It is an object of the invention to provide a kind of Vitamin C Two-step Fermentation method, this method is by improving fermentation medium
Formula, optimization for fermentation technology, so as to effectively increase rate of producing acid, improves Cologne acid yield, improves fermentation conversion rate, and
Material residual during fermentation ends can be effectively reduced, improves zymotic fluid quality.
The technical solution taken for achieving the above object is：
A kind of Vitamin C Two-step Fermentation production method, including it is mixed to being made of bacillus megaterium and raw ketone group 2-KLG bacterium
The three-level seed for closing fungus strain expands culture, it is characterized in that, cultured three-level seed liquor is directly connected to what sterilization treatment was crossed
Fermented and cultured is carried out in fermentation medium, while stream adds L- sorboses at the very start in fermentation,
Wherein described fermentation medium, which forms, is：Urea 0.1~0.3%, biotin 0.01~0.04%, corn pulp 1.5~2.5%,
Potassium dihydrogen phosphate 0.1~0.3%, magnesium sulfate 0.01~0.04%.
The fermentation culture conditions are：Inoculum concentration 15~30%, 28~30 DEG C of cultivation temperature；Throughput 1：0.5~1.2v/
V/min, stirring, feed supplement；Fermentation process dissolved oxygen ensures that more than 20% 40~45hr of cultivation cycle puts tank.
The stream dosage of the L- sorboses is to control in zymotic fluid on the basis of 15~35mg/ml of residual sugar, and 4~8h is tied before putting tank
Line adds L- sorboses, puts tank residual sugar and is less than 0.6mg/ml.
Also need to mend lye during the fermentation, the lye is that mass concentration is 25% sterile sodium carbonate solution, control hair
PH7.0~7.5 in zymotic fluid.
The three-level seed expands in culture
Primary-seed medium forms：L- sorboses 1.2~1.5%, glucose 0.2~0.5%, magnesium sulfate 0.08~0.12%,
Urea 0.1~0.2%, corn pulp 1.0~1.2%, calcium carbonate 0.05~0.1%；PH 7.0~7.4；
First order seed incubation controls：Cultivation temperature：28~30 DEG C, throughput 1：1.0~1.5v/v/min is simultaneously stirred, culture
Cycle：24±4h；
Secondary seed medium forms：L- sorboses 1.2~1.5%, glucose 0.2~0.5%, magnesium sulfate 0.08~0.12%,
Urea 0.1~0.2%, corn pulp 1.0~1.2%, calcium carbonate 0.05~0.1%；PH 7.0~7.4；
Secondary seed incubation controls：Inoculum concentration：3~5%；Cultivation temperature：28~30 DEG C；Throughput 1：1.0~1.5v/v/
Min is simultaneously stirred, cultivation cycle：20~28hr；
Three-level seed culture medium forms：L- sorboses 1.2~1.5%, glucose 0.2~0.5%, magnesium sulfate 0.01~0.04%,
Urea 0.1~0.2%, corn pulp 1.0~1.5%, calcium carbonate 0.08~0.20%；PH 7.0~7.4；
Three-level seed culture process control：Inoculum concentration：8~10%；Cultivation temperature：28~30 DEG C；Throughput 1：1.0~1.5v/v/
Min is simultaneously stirred, cultivation cycle：16~24h.
Using the above method, the tank cycle is finally put：40~45h；Put 143.3~152.4mg/ml of tank acid content；Residual sugar≤
0.6mg/ml；Filtrate light transmittance：90.7~92.3%；Fermentation index：3.47~3.73g/L.h；Average rate of producing acid 3.342~
3.676 mg/ml.h。
The present invention's has the technical effect that the following aspects：
1st, present invention optimizes fermentative medium formula, biotin is added in the medium, reduces the use of corn pulp.It is beautiful
Rice ＆ peanut milk is one of component of fermentation medium, is made with the soak water of maize in corn starch or sugaring.Appearance color is in deep brown
Color, Solid content or uniform and smooth are rough irregular；Corn pulp is because it is rich in soluble protein and a variety of free amino acids and in VC
Belong to main nitrogen in two-step fermentation production, but its Solid content amount number, the fineness degree of Solid content can influence fermenting and producing
Stabilization, and the color and luster of fermentation liquid can be deepened, the extraction, crystallization to lower procedure impact.So add biotin
And the dosage of corn pulp is reduced to optimize fermented and cultured environment, it can preferably promote the coordination between acid-producing bacteria and concomitance bacterium and make
It reaches optimum state, improves rate of producing acid, improves Cologne acid yield, improves fermentation conversion rate conversion ratio, reduces fermentation ends
When material residual, improve zymotic fluid quality.By above-mentioned change, zymotic fluid appearance luster is good, puts tank filtrate light transmittance and carries
It is high by more than 5%.
2nd, the present invention is the conjunction sugar operation deleted after sterilizing in fermentation medium, reduces after shifting sorbose in nutrient solution
Concentration, reduce the osmotic pressure of thalli growth environment；Using stream adds the feeding strategy of sorbose immediately after culture transferring, height is relieved
Concentration of substrate improves mixed bacterium growth metabolism environment, fermentation period is shortened 5~10h to the reptation behavior of thalli growth.
3rd, by the addition combined formulation of biotin and the optimization of supplying technics, effectively improve the growth of mixed bacterium environment,
Thalline quality is improved, promotes growth, propagation, metabolism and the production acid of small bacterium；2-KLG be averaged rate of producing acid improve 10% with
On；Fermentation index improves more than 10%.
Embodiment
The present invention is explained with example, it should be understood that example is to be used to illustrate rather than to this below
The limitation of invention.The scope of the present invention is determined with core content according to claims.
In following comparative examples and embodiment, fermenting microbe is bacillus megaterium (being commonly called as big bacterium) and ordinary student ketone group
The mixed culture of 2-KLG bacterium (being commonly called as small bacterium) composition.
Comparative example
1 actication of culture：Strain one will be taken according to a conventional method, be accessed by certain inoculum concentration in shake-flask seed culture medium, 180~
220rpm, 29 ± 1 DEG C of 20 ± 4h of constant-temperature shaking culture, to Cologne acid content >=5mg/ml, three generations is obtained after activating three generations by this
Kind liquid, three generations's kind liquid is inoculated in the seed culture medium to have sterilized by 2~10% inoculum concentration.
2 three-level seeds expand culture：
2.1 first order seed：
Primary-seed medium（%）：L- sorboses 1.2, glucose 0.25, magnesium sulfate 0.09, urea 0.13, corn pulp 1.0, carbon
Sour calcium 0.08；7.0~7.4,118~121 DEG C of 15~20min of sterilizing of pH.
Process control：Bacillus megaterium (being commonly called as big bacterium) and ordinary student ketone group 2-KLG bacterium (being commonly called as small bacterium) are formed
Mixed culture is accessed in above-mentioned first cell culture medium, the strain prepared is accessed in above-mentioned first cell culture medium, inoculum concentration：5%, training
Support temperature：28~30 DEG C；Throughput 1：1.0~1.5v/v/min is simultaneously stirred, cultivation cycle：24±2hr.
2.2 secondary seed：
Secondary seed medium（%）：L- sorboses 1.2, glucose 0.25, magnesium sulfate 0.09, urea 0.13, corn pulp 1.0, carbon
Sour calcium 0.08；7.0~7.4,118~121 DEG C of 15~20min of sterilizing of pH.
Process control：Above-mentioned 2.1 gained primary seed solution is accessed in secondary seed medium, inoculum concentration：5%；Culture temperature
Degree：28~30 DEG C, throughput 1：1.0~1.5v/v/min is simultaneously stirred, cultivation cycle：24±2hr.
2.3 three-level seeds：
Three-level seed culture medium（%）：L~sorbose 1.5, glucose 0.25, magnesium sulfate 0.09, urea 0.14, corn pulp 1.1,
Calcium carbonate 0.08；PH7.0~7.4,118~121 DEG C of 15~20min of sterilizing.
Process control：Above-mentioned 2.2 gained secondary seed solution is accessed in three-level seed culture medium, inoculum concentration：10%；Culture
Temperature：28~30 DEG C；Throughput 1：1.0~1.5v/v/min is simultaneously stirred, cultivation cycle：20±4hr.
3 fermentations：
3.1 fermentation medium（%）：L- sorboses：8.0th, urea 0.14, corn pulp 4.0, potassium dihydrogen phosphate 0.15, magnesium sulfate
0.015；7.0~7.4,118~121 DEG C of 15~20min of sterilizing of pH.
3.2 ferment control：
Close sugar：L- sorboses are incorporated into by 40~50% concentration after fermentation medium sterilizing.
Above-mentioned 2.3 gained three-level seed liquor is accessed in fermentation medium, inoculum concentration：25%；Cultivation temperature：28~30 DEG C；
Throughput 1：0.5~1.2v/v/min, stirring, feed supplement, fermentation process dissolved oxygen ensure more than 20%；Cultivation cycle：50h puts tank.
3.3 process feed supplements：(carbon source, lye are filled into fermentation process)
Carbon source：That is L- sorboses, are the products of VC one-step fermentation sorbierites, supply growth, the propagation of Gluconobacter oxvdans
And the conversion and production of 2-keto-L-gulonic acid.
Lye：That is 25% sterile sodium carbonate solution, the conditioning agent as VC two-step fermentation process nutrient solutions pH.
Mend alkali control：When PH drops to 6.5 after culture transferring, start stream plus 25% sterile sodium carbonate solution, adjusted with pH value plus alkali
Amount, makes pH value be controlled by fermentation period gradient 7.0~7.5.
Mend sugar control：Fermented and cultured about 4~7h starts stream plus L- sorboses, and fermentation process is with residual sugar（I.e.：Fermentation culture
The concentration of middle L- sorboses）Adjust and mend sugar amount, residual sugar control range is in 12~25mg/ml, and 4~8h terminates sorbose before tank is put
Stream adds, and puts tank residual sugar and is less than 0.6mg/ml.
3.4 put tank parameter：
Put the tank cycle：50hr；Cologne acid content：132.9mg/ml；Residual sugar：0.42mg/ml；Filtrate light transmittance：84.2%；It is average
Rate of producing acid：2.658mg/ml.h；Fermentation index 3.026g/L.h.
Embodiment 1
1 actication of culture：With the actication of culture in comparative example.
2 three-level seeds expand culture：Expand culture with three-level seed in comparative example.
3 fermentations：
3.1 fermentation medium（%）：Urea 0.12, corn pulp 2.5, potassium dihydrogen phosphate 0.16, magnesium sulfate 0.015, biotin
0.015；7.0~7.4,118~121 DEG C of 15~20min of sterilizing of pH.
3.2 ferment control：Gained three-level seed liquor is directly accessed in fermentation medium, while is flowed at the very start in fermentation
Add L- sorboses.
Inoculum concentration：20%th, cultivation temperature：28~30 DEG C, throughput 1：0.5~1.2v/v/min, stirring, feed supplement, fermented
Journey dissolved oxygen ensures more than 20%；Cultivation cycle：45h puts tank.
Stream plus during sorbose, controls zymotic fluid 15~35mg/ml of residual sugar, and 4~8h terminates sorbose stream and adds before tank is put, and puts
Tank residual sugar is less than 0.6mg/ml.
3.3 process feed supplements：(lye is filled into fermentation process)
Lye and benefit lye control same comparative example.
3.4 put tank parameter：
Put the tank cycle：45hr；Cologne acid content：143.8mg/ml；Residual sugar：0.51mg/ml；Filtrate light transmittance：90.7%；It is average
Rate of producing acid：3.196mg/ml.h；Fermentation index 3.3432g/L.h.
Embodiment 2
1 actication of culture：With the actication of culture in comparative example.
2 three-level seeds expand culture：Expand culture with three-level seed in comparative example.
3 fermentations：
3.1 fermentation medium（%）：Urea 0.14, corn pulp 2.0, potassium dihydrogen phosphate 0.16, magnesium sulfate 0.015, biotin
0.025；7.0~7.4,118~121 DEG C of 15~20min of sterilizing of pH.
3.2 ferment control：Gained three-level seed liquor is directly accessed in fermentation medium, while is flowed at the very start in fermentation
Add L- sorboses.
Inoculum concentration：20%；Cultivation temperature：28~30 DEG C；Throughput 1：0.5~1.2v/v/min, stirring, feed supplement, fermented
Journey dissolved oxygen ensures more than 20%, cultivation cycle：45hr puts tank.
Stream plus during sorbose, controls zymotic fluid 15~35mg/ml of residual sugar, and 4~8h terminates sorbose stream and adds before tank is put, and puts
Tank residual sugar is less than 0.6mg/ml.
3.3 process feed supplements：(lye is filled into fermentation process)
Lye and benefit lye control same comparative example.
3.4 put tank parameter：
Put the tank cycle：45hr；Cologne acid content：152.4mg/ml, residual sugar 0.36mg/ml, filtrate light transmittance：91.2%；Average production
Mollic acid：3.387mg/ml.h；Fermentation index 3.395g/L.h.
Embodiment 3
1 actication of culture：With the actication of culture in comparative example.
2 three-level seeds expand culture：Expand culture with three-level seed in comparative example.
3 fermentations：
3.1 fermentation medium（%）：Urea 0.14, corn pulp 1.5, potassium dihydrogen phosphate 0.18, magnesium sulfate 0.015, biotin 0.04
；7.0~7.4,118~121 DEG C of 15~20min of sterilizing of pH.
3.2 ferment control：Gained three-level seed liquor is directly accessed in fermentation medium, while is flowed at the very start in fermentation
Add L- sorboses.
Inoculum concentration：30%；Cultivation temperature：28~30 DEG C；Throughput 1：0.5~1.2v/v/min, stirring, feed supplement, fermented
Journey dissolved oxygen ensures more than 20%, cultivation cycle：40hr puts tank.
Stream plus during sorbose, controls zymotic fluid 15~35mg/ml of residual sugar, and 4~8h terminates sorbose stream and adds before tank is put, and puts
Tank residual sugar is less than 0.6mg/ml.
3.3 process feed supplements：(lye is filled into fermentation process)
Lye and benefit lye control same comparative example.
3.4 put tank parameter：
Put the tank cycle：40hr；Cologne acid content：153.6mg/ml, residual sugar 0.46mg/ml, filtrate light transmittance：92.3%；Average production
Mollic acid：3.583mg/ml.h；Fermentation index 3.676g/L.h.
The above is only the preferred embodiment of the present invention, and the present invention can provide a kind of way of composing, to different bacterium
The concomitance bacterium of category can also make corresponding adjustment on the premise of this design, these adjustment also should be regarded as the protection model of the present invention
Enclose.
Claims (5)
1. a kind of Vitamin C Two-step Fermentation production method, including to being made of bacillus megaterium and raw ketone group 2-KLG bacterium
The three-level seed of mixed culture expands culture, it is characterized in that, cultured three-level seed liquor is directly connected to sterilization treatment mistake
Fermentation medium in carry out fermented and cultured, while in fermentation stream plus L~sorbose at the very start,
Wherein described fermentation medium, which forms, is：Urea 0.1~0.3%, biotin 0.01~0.04%, corn pulp 1.5~2.5%,
Potassium dihydrogen phosphate 0.1~0.3%, magnesium sulfate 0.01~0.04%.
2. Vitamin C Two-step Fermentation production method described in accordance with the claim 1, it is characterised in that the fermentation culture conditions
For：Inoculum concentration 15~30%, 28~30 DEG C of cultivation temperature；Throughput 1：0.5~1.2v/v/min, stirring, feed supplement；Fermentation process
Dissolved oxygen ensures that more than 20% 40~45hr of cultivation cycle puts tank.
3. Vitamin C Two-step Fermentation production method described in accordance with the claim 1, it is characterised in that the stream of the L- sorboses adds
For amount to control in zymotic fluid on the basis of 15~35mg/ml of residual sugar, 4~8h terminates stream plus L- sorboses before putting tank, and it is low to put tank residual sugar
In 0.6mg/ml.
4. Vitamin C Two-step Fermentation production method described in accordance with the claim 1, it is characterised in that also need during the fermentation
Lye is mended, the lye is that mass concentration is 25% sterile sodium carbonate solution, controls pH7.0~7.5 in zymotic fluid.
5. Vitamin C Two-step Fermentation production method described in accordance with the claim 1, it is characterised in that the three-level seed expands training
In supporting
Primary-seed medium forms：L- sorboses 1.2~1.5%, glucose 0.2~0.5%, magnesium sulfate 0.08~0.12%,
Urea 0.1~0.2%, corn pulp 1.0~1.2%, calcium carbonate 0.05~0.1%；PH 7.0~7.4；
First order seed incubation controls：Cultivation temperature：28~30 DEG C, throughput 1：1.0~1.5v/v/min is simultaneously stirred, culture
Cycle：24±4h；
Secondary seed medium forms：L- sorboses 1.2~1.5%, glucose 0.2~0.5%, magnesium sulfate 0.08~0.12%,
Urea 0.1~0.2%, corn pulp 1.0~1.2%, calcium carbonate 0.05~0.1%；PH 7.0~7.4；
Secondary seed incubation controls：Inoculum concentration：3~5%；Cultivation temperature：28~30 DEG C；Throughput 1：1.0~1.5v/v/
Min is simultaneously stirred, cultivation cycle：20~28hr；
Three-level seed culture medium forms：L- sorboses 1.2~1.5%, glucose 0.2~0.5%, magnesium sulfate 0.01~0.04%,
Urea 0.1~0.2%, corn pulp 1.0~1.5%, calcium carbonate 0.08~0.20%；PH 7.0~7.4；
Three-level seed culture process control：Inoculum concentration：8~10%；Cultivation temperature：28~30 DEG C；Throughput 1：1.0~1.5v/v/
Min is simultaneously stirred, cultivation cycle：16~24h.
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| CN112301071A (en) * | 2020-11-04 | 2021-02-02 | 赤峰蒙广生物科技有限公司 | Method for producing adenine by fermentation method |
| CN114292893A (en) * | 2022-01-05 | 2022-04-08 | 山东天力药业有限公司 | Method for adding multiple trace elements into VC mixed bacteria seed tank to shorten fermentation period and improve yield of acid production |
| CN114292893B (en) * | 2022-01-05 | 2023-08-29 | 山东天力药业有限公司 | Method for shortening fermentation period and improving acid yield by adding various microelements into VC mixed strain seed tank |
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