CN106434830A - Method for improving 2-keto-L-gluconic acid fermentation efficiency - Google Patents
Method for improving 2-keto-L-gluconic acid fermentation efficiency Download PDFInfo
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- CN106434830A CN106434830A CN201611180685.8A CN201611180685A CN106434830A CN 106434830 A CN106434830 A CN 106434830A CN 201611180685 A CN201611180685 A CN 201611180685A CN 106434830 A CN106434830 A CN 106434830A
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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
The invention relates to a method for improving 2-keto-L-gluconic acid fermentation efficiency. The method is characterized in that in the process of mixed strains fermenting 2-keto-L-gluconic acid, the mixed strains comprise ketogulonigenium vulgare and bacillus megaterium, seed solution cultured to the middle and later periods of logarithmic growth is inoculated into fermentation tank optimum culture media to ferment and culture, sorbose is fed since fermentation time is 0h, and small material culture media are fed at the prior period of the logarithmic growth. According to the method, production intensity of the 2-keto-L-gluconic acid can be improved, efficiency is improved, production capacity is optimized, the content of the 2-keto-L-gluconic acid in fermentation solution can reach 115.6-150.3mg/ml, acid producing rate is 2.89-3.76g.(L.h)-1, and the conversion rate of glucose acid is 87.2-95.0%.
Description
Technical field
The invention belongs to technical field of microbial fermentation, more particularly to a kind of raising KGA fermentation life
The method producing efficiency, is partial to transform Vitamin C Two-step Fermentation technique.
Background technology
Vitamin C two step mixed fungus fermentation system, including two fermentation steps, claims two-step fermenting：The first step is in black vinegar
Under acidfast bacilli effect, D-glucitol is oxidized to L- sorbose, is commonly called as alcohol sugar conversion；This step fermentation technology maturation and conversion ratio
Height, can reach more than 98%；Clearer to its related gene and conversion base Quality Research both at home and abroad at present；Second step
It is that L- sorbose is further oxidized to KGA, be commonly called as saccharic acid conversion；Second step is that the mixing of two kinds of bacterium is sent out
Ferment, L- sorbose ordinary student ketone group 2-KLG bacterium and bacillus megaterium composition mixed culture effect under, by biological oxidation
Become KGA, raw ketone group 2-KLG bacterium is that acid-producing bacteria is commonly called as little bacterium, and bacillus megaterium is that concomitance bacterium is commonly called as greatly
Bacterium, during mixed fermentation, concomitance bacterium grows in advance, and metabolism discharges the albumen with biological activity, acts on acid-producing bacteria therewith, opens
Move its growth, produce acid.I.e. the growth of acid-producing bacteria and acid producing ability are largely restricted by concomitance bacterium growth metabolism, therefore logical
Cross and improve original production technology, improve big bacteria growing environment, adjust big bacterium physiological statuss, promote mixed bacterium metabolism, for raising 2-
KLG fermentation production efficiency, raising production capacity, reduction production cost have conclusive effect.
Content of the invention
The present invention be directed in existing vitamin C producing process, the low problem of second step fermentation production efficiency, in providing
A kind of production efficiency changing into KGA by improving L- sorbose, and then improve 2- keto-L-gulonic
The method of acid fermentation production efficiency.
As a rule, as a key factor in industrial fermentation processes, it is related to holding of lag phase to vaccination ways
The aspects such as the yield of the continuous, accumulation of the growth rate of logarithmic (log) phase, biomass and end-product.
Based on this, set up technical scheme, specially：
A kind of method improving KGA fermentation production efficiency, is characterized in that：With ordinary student ketone group 2-KLG bacterium
In the sweat of the mixed culture fermenting and producing KGA of bacillus megaterium composition, will cultivate to logarithm
The intermediary and later stages seed liquor of trophophase is seeded in fermentation tank Optimal Medium and carries out fermentation culture；Meanwhile, start to flow from fermentation 0h
Plus sorbose, and add and fill into small powder culture medium in fermentation logarithmic growth early stage stream；
Described fermentation tank Optimal Medium consists of：Carbamide 0.05～0.10, Semen Maydis pulp 0.5～1.0, potassium dihydrogen phosphate 0.02
～0.05, magnesium sulfate 0.01, Calcium Carbonate 0.05, pH 6.7～7.4, by weight percentage；
Described small powder culture medium consists of：Sorbose 5.0, carbamide 0.1～0.2, Semen Maydis pulp 2.0～5.0, potassium dihydrogen phosphate
0.02～0.05, Calcium Carbonate 0.05, pH 6.7～7.4, by weight percentage.
The seed liquor of described middle logarithmic growth intermediary and later stages refers to cultivate seed liquor to exponential phase 20 ± 5h, GULONG
It is seeded to during acid content >=5mg/ml in fermentation tank Optimal Medium, now can obtain the vigorous thalline of vitality, also may be used simultaneously
Guarantee to obtain higher cell concentration, be conducive to the shortening of the period of delay of thalli growth in sweat.
In sweat, control sorbose concentration 12～35mg/ml, put tank residual sugar and be less than 0.6mg/ml, 4 before putting tank～
The stream that 8h terminates sorbose adds.
In sweat, small powder culture medium stream totalling amount be inoculation after fermentation liquid amass 10～20%, and 10h ~
Terminate stream in 15h to add.
In sweat, stream Jia 20～25% sterile sodium carbonate solution regulation fermentation liquid pH value between 6.7～7.4.
Sorbose in described small powder culture medium is separately sterilized with other raw materials, is cooled to less than 40 DEG C and mixes, and controls little
In material culture medium, initial sorbose concentration is 25～40mg/ml.
The present invention is the mixed culture production 2- ketone group-L- in ordinary student ketone group 2-KLG bacterium and bacillus megaterium composition
Optimize fermentation medium during 2-KLG, its object is to：Reduce thalli growth ring by reducing fermentation basestocks concentration
The osmotic pressure in border；The inhibition releasing high concentration substrate to thalli growth, so that thalline increases rapidly in suitable environment, contracts
Short earlier fermentation required time；Overcome and cause cell raised growth, oxygen consumption excessive because of substrate concentration height, so that ventilation（Stirring）If
The standby situation that can not mate, to improve mixed bacteria growing metabolism environment.Specifically by culture to the logarithmic growth intermediary and later stages
Seed liquor moves in optimization fermentation medium, starts stream plus mend sorbose from 0h, and is added with flowing in fermentation logarithmic growth early stage
Mode fill into small powder terminate in 10-15h stream plus, to stimulate big bacteria growing, shorten its lag phase, extend its balance period, be
The growth and breeding of little bacterium and product acid provide more excellent environment-guarantee.KGA can be lifted by the present invention to produce
Intensity, the efficiency that improves, optimization production capacity.By the method for the present invention, KGA content in fermentation liquid can be made to reach
115.6～150.3mg/ml, rate of producing acid 2.89～3.76g. (L.h) -1, saccharic acid conversion ratio is 87.2～95%.
Specific embodiment
It is explained the present invention with example below it should be understood that example is for illustrating rather than to this
The restriction of invention.The scope of the present invention is determined according to claims with core content.
In following reference examples and embodiment, strain is ordinary student ketone group 2-KLG bacterium and the mixing of bacillus megaterium composition
Fungus strain.
Reference examples：
1 strain preparation：Strain one will be taken according to a conventional method, access in shake-flask seed culture medium by certain inoculum concentration, 180～
220rpm, 29 ± 1 DEG C of constant-temperature shaking culture 20 ± 4h, to GULONG acid content >=5mg/ml, obtain three generations by after this activation three generations
Plant liquid, three generations's kind liquid is inoculated in the seed culture medium having sterilized by 2～10% inoculum concentration.
2 kinds of liquid preparations：
2.1 seed tank culture based formulas (%)：L- sorbose 1.5, glucose 0.2, magnesium sulfate 0.02, yeast extract 0.5, carbamide
0.4th, Semen Maydis pulp 0.3, Calcium Carbonate 0.4, potassium dihydrogen phosphate 0.5, pH 6.7～7.4.
2.2 seed tank culture conditions：28～30 DEG C of cultivation temperature, ventilation ratio 1：1.0～1.5v/v/min simultaneously stirs, culture
During control dissolved oxygen 30～50%, cultivate to exponential phase 20 ± 5hr, GULONG acid content >=5mg/ml.
3 fermentations：
3.1 fermentor cultivation based formulas (%)：Sorbose 8.0, carbamide 0.1, Semen Maydis pulp 2.0, potassium dihydrogen phosphate 0.05, sulphuric acid
Magnesium 0.01, Calcium Carbonate 0.05, pH 6.7～7.4.
3.2 fermentation tank culture medium sterilization methods：Sorbose is separately sterilized with other raw materials, is cooled to less than 40 DEG C and mixes
Close, and make fermentation medium initial sorbose concentration be 20～40mg/ml.
3.3 fermentation tank vaccination ways：Treat that kind of liquid is cultivated to exponential phase 20 ± 5hr, GULONG acid content >=5mg/ml, mirror
Inspection no miscellaneous bacteria when, by 20～40% inoculum concentration move into fermentation medium in.
3.4 fermentor cultivation conditions：
28～31 DEG C of cultivation temperature, ventilation 1：0.5～1.2v/v/min, stirring, feed supplement, sweat ensures that sweat is molten
Oxygen more than 20%, culture 40hr puts tank.
3.5 sweat feed supplements：Concentration, in 12～35mg/ml, is put tank residual sugar and is less than 0.6mg/ml, according to fermentation culture
The concentration of middle L- sorbose, before putting tank, 4～8h stream plus L- sorbose terminate；Sweat stream adds 20～25% sterile sodium carbonate
Solution, adjusts pH value between 6.7～7.4.
3.6 fermentation culture terminals：KGA concentration is 111.6g.L-1, fermentation period 40h, rate of producing acid
2.79g. (L.h) -1, saccharic acid conversion ratio is 87.8%.
Embodiment 1：
1 strain preparation, the kind same reference examples of liquid preparation process.
2 fermentations：
2.1 fermentation tanks optimization culture based formulas (%)：Carbamide 0.05～0.10, Semen Maydis pulp 0.5～1.0, potassium dihydrogen phosphate 0.02
～0.05, magnesium sulfate 0.01, Calcium Carbonate 0.05, pH 6.7～7.4.
2.2 small powder culture medium prescriptions (%)：Sorbose 5.0, carbamide 0.1～0.2, Semen Maydis pulp 2.0～5.0, biphosphate
Potassium 0.02～0.05, Calcium Carbonate 0.05, pH 6.7～7.4.
2.3 small powder medium sterilization modes：L- sorbose is separately sterilized with other raw materials, is cooled to less than 40 DEG C and mixes
Close, and make initial sorbose concentration be 25～40mg/ml.
2.4 fermentation tank vaccination ways and condition of culture：Same reference examples.
2.5 sweat feed supplements：Fermentation 0h plays stream plus sorbose, and cocurrent adds benefit small powder and terminates in 10h, and small powder fills into always
Measure for connect rear volume 10～20%；Sweat sorbose concentration controls in 12～35mg/ml, puts tank residual sugar and is less than 0.6mg/
Ml, according to the concentration of L- sorbose in fermentation culture, before putting tank, 4～8h stream plus L- sorbose terminate；Sweat stream adds
20～25% sterile sodium carbonate solution adjust pH value between 6.7～7.4.
2.6 fermentation termination：KGA concentration is 126.81g.L-1, fermentation period 40h, rate of producing acid
3.17g. (L.h) -1, saccharic acid conversion ratio is 89.4%.
Embodiment 2：
1 strain preparation, the kind same reference examples of liquid preparation process.
2 fermentations：
2.1 fermentation tanks optimization culture based formulas (%), small powder culture medium prescription (%) and sterilization method：With embodiment 1.
2.2 fermentation tank vaccination ways and condition of culture：Same reference examples.
2.3 sweat feed supplements：Fermentation 0h plays stream plus sorbose, and cocurrent adds benefit small powder and flows in 12h plus terminate, and small powder is mended
Entering total amount is connect rear volume 10～20%；Sweat sorbose concentration controls in 12～35mg/ml, puts tank residual sugar and is less than
0.6mg/ml, according to the concentration of L- sorbose in fermentation culture, before putting tank, 4～8h stream plus L- sorbose terminate；Fermented
Journey stream adds 20-25% sterile sodium carbonate solution and adjusts pH value between 6.7～7.4.
2.4 fermentation termination：KGA concentration is 133g.L-1, fermentation period 40h, rate of producing acid 3.33g.
(L.h) -1, saccharic acid conversion ratio is 91.8%.
Embodiment 3：
1 strain preparation, the kind same reference examples of liquid preparation process.
2 fermentations：
2.1 fermentation tanks optimization culture based formulas (%), small powder culture medium prescription (%) and sterilization method：With embodiment 1.
2.2 fermentation tank vaccination ways and condition of culture：Same reference examples.
2.3 sweat feed supplements：Fermentation 0h plays stream plus sorbose, and cocurrent adds benefit small powder and flows in 15h plus terminate, and small powder is mended
Entering total amount is connect rear volume 10～20%；Sweat sorbose concentration controls in 12～35mg/ml, puts tank residual sugar and is less than
0.6mg/ml, according to the concentration of L- sorbose in fermentation culture, before putting tank, 4～8h stream plus L- sorbose terminate；Fermented
Journey stream adds 20～and 25% sterile sodium carbonate solution adjusts pH value between 6.7～7.4.
2.4 fermentation termination：KGA concentration is 150.3g.L-1, fermentation period 40h, rate of producing acid
3.76g. (L.h) -1, saccharic acid conversion ratio is 95.0%.
Embodiment 4：
1 strain preparation, the kind same reference examples of liquid preparation process.
2 fermentations：
2.1 fermentation tanks optimization culture based formulas (%), small powder culture medium prescription (%) and sterilization method：With embodiment 1.
2.2 fermentation tank vaccination ways and condition of culture：Same reference examples.
2.3 sweat feed supplements：Fermentation 0h plays stream plus sorbose, and cocurrent adds benefit small powder and flows in 20h plus terminate, and small powder is mended
Entering total amount is connect rear volume 10～20%；Sweat sorbose concentration controls in 12～35mg/ml, puts tank residual sugar and is less than
0.6mg/ml, according to the concentration of L- sorbose in fermentation culture, before putting tank, 4～8h stream plus L- sorbose terminate；Fermented
Journey stream adds 20～and 25% sterile sodium carbonate solution adjusts pH value between 6.7～7.4.
2.4 fermentation termination：KGA concentration is 115.6g.L-1, fermentation period 40h, rate of producing acid
2.89g. (L.h) -1, saccharic acid conversion ratio is 87.2%.
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 belonging to can also make corresponding adjustment on the premise of this design, and these adjustment also should be regarded as the protection model of the present invention
Enclose.
Claims (6)
1. a kind of method improving KGA fermentation production efficiency, is characterized in that：With ordinary student ketone group 2-KLG
In the sweat of mixed culture fermenting and producing KGA of bacterium and bacillus megaterium composition, will cultivate to right
The intermediary and later stages seed liquor of number trophophase is seeded in fermentation tank Optimal Medium and carries out fermentation culture；Meanwhile, from fermentation 0h
Stream plus sorbose, and add and fill into small powder culture medium in fermentation logarithmic growth early stage stream；
Described fermentation tank Optimal Medium consists of：Carbamide 0.05～0.10, Semen Maydis pulp 0.5～1.0, potassium dihydrogen phosphate 0.02
～0.05, magnesium sulfate 0.01, Calcium Carbonate 0.05, pH 6.7～7.4, by weight percentage；
Described small powder culture medium consists of：Sorbose 5.0, carbamide 0.1～0.2, Semen Maydis pulp 2.0～5.0, potassium dihydrogen phosphate
0.02～0.05, Calcium Carbonate 0.05, pH 6.7～7.4, by weight percentage.
2. according to the raising KGA fermentation production efficiency described in claim 1 method it is characterised in that institute
The seed liquor stating the middle logarithmic growth intermediary and later stages refers to cultivate seed liquor to exponential phase 20 ± 5h, and GULONG acid content >=
It is seeded to during 5mg/ml in fermentation tank Optimal Medium.
3. according to the method improving KGA fermentation production efficiency described in claim 1 it is characterised in that sending out
During ferment, control sorbose concentration 12～35mg/ml, put tank residual sugar and be less than 0.6mg/ml, 4～8h terminates Pyrusussuriensiss before putting tank
The stream of sugar adds.
4. according to the method improving KGA fermentation production efficiency described in claim 1 it is characterised in that sending out
During ferment, the stream totalling amount of small powder culture medium is long-pending 10～20% of inoculation after fermentation liquid, and terminates in 10h ~ 15h
Stream adds.
5. according to described in claim 1 according to the raising KGA fermentation production efficiency described in claim 1
It is characterised in that in sweat, stream Jia 20 to method～25% sterile sodium carbonate solution regulation fermentation liquid pH value is 6.7～7.4
Between.
6. according to described in claim 1 according to the raising KGA fermentation production efficiency described in claim 1
Method, it is characterised in that the sorbose in described small powder culture medium is separately sterilized with other raw materials, is cooled to less than 40 DEG C and mixes,
Initial sorbose concentration in small powder culture medium is controlled to be 25～40mg/ml.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955828A (en) * | 2017-11-28 | 2018-04-24 | 宁夏启元药业有限公司 | A kind of Vitamin C Two-step Fermentation production method |
| CN108796025A (en) * | 2018-06-11 | 2018-11-13 | 石药集团维生药业（石家庄）有限公司 | A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique |
| CN109593813A (en) * | 2018-12-28 | 2019-04-09 | 黑龙江新和成生物科技有限公司 | The fermentation culture medium of 2-keto-L-gulonic acid and the fermentation method for producing of 2-keto-L-gulonic acid |
| CN110760468A (en) * | 2019-11-29 | 2020-02-07 | 宁夏启元药业有限公司 | Method for improving efficiency of vitamin C precursor 2-keto-L-gulonic acid |
| 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 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107955828A (en) * | 2017-11-28 | 2018-04-24 | 宁夏启元药业有限公司 | A kind of Vitamin C Two-step Fermentation production method |
| CN108796025A (en) * | 2018-06-11 | 2018-11-13 | 石药集团维生药业（石家庄）有限公司 | A kind of improved 2-keto-L-gulonic acid fermentation manufacturing technique |
| CN109593813A (en) * | 2018-12-28 | 2019-04-09 | 黑龙江新和成生物科技有限公司 | The fermentation culture medium of 2-keto-L-gulonic acid and the fermentation method for producing of 2-keto-L-gulonic acid |
| CN109593813B (en) * | 2018-12-28 | 2021-06-15 | 黑龙江新和成生物科技有限公司 | Culture medium for fermentation of 2-keto-L-gulonic acid and fermentation production method of 2-keto-L-gulonic acid |
| CN110760468A (en) * | 2019-11-29 | 2020-02-07 | 宁夏启元药业有限公司 | Method for improving efficiency of vitamin C precursor 2-keto-L-gulonic acid |
| 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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