CN109593813A - The fermentation culture medium of 2-keto-L-gulonic acid and the fermentation method for producing of 2-keto-L-gulonic acid - Google Patents
The fermentation culture medium of 2-keto-L-gulonic acid and the fermentation method for producing of 2-keto-L-gulonic acid Download PDFInfo
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Abstract
The present invention provides a kind of fermentation culture medium of 2-keto-L-gulonic acid, contain the mixotrophism agent of 0.007~0.009g/L, the mixotrophism agent weight percent composition are as follows: 25~67% cromoci and 33~75% ferroheme.The present invention also provides a kind of fermentation method for producing of 2-keto-L-gulonic acid, the mixotrophism agent is added in supplement when being down to 0.001~0.003g/L by mixotrophism agent concentration in the fermentation medium, to maintain its concentration in 0.001~0.003g/L until fermentation ends, or when online oxygen dissolving value is 20%~40%, supplement is added pyrroloquinoline quinone and makes its 0.003~0.005g/L of concentration in the medium, the combined coefficient that can shorten fermentation period, improve 2-keto-L-gulonic acid.
Description
Technical field
The invention belongs to technical field of microbial fermentation, more particularly it relates to which a kind of novel 2- ketone group-L- is ancient
The fermentation culture medium of imperial acid and the fermentation method for producing of 2-keto-L-gulonic acid, use the culture medium and fermentation method for producing
The combined coefficient of 2-keto-L-gulonic acid can be improved, shorten fermentation period.
Background technique
Vitamin C (Vitamin C, abbreviation VC), i.e. L-AA (L-Ascorbic acid) are that human body must be from
A kind of water soluble vitamin of external world's intake, is widely used in fields such as medicine, food, cosmetics, feeds.2- ketone group-L- is ancient
Imperial acid (2-keto-L-gulonic acid, abbreviation 2-KGA) is the ascorbic important as precursors of synthesis, current " two-step fermenting "
It is still the main technique of industrialized production VC, wherein second step mixed fungus fermentation is the key technology of the technique, which only has
Big bacterium (or concomitance bacterium) nutrition auxiliary under, small bacterium can normal growth metabolism, synthesize relevant enzyme.Secondly, small bacterium benefit
Sorb ketone is converted by sorbose with the sorbose dehydrogenase on cell membrane, then by sorb ketone under sorbic ketone dehydrogenase effect
It is converted into 2-KGA, while the electronics taken off from sorbose and sorb ketone is passed into oxygen through the electron transport chain on cell membrane
Negative oxygen ion is formed, negative oxygen ion ultimately generates water in conjunction with the hydrogen ion from sorbose and sorb ketone.Therefore in the hair
In ferment technique, the efficiency by improving enzyme activity and electron transmission is very important to improve the combined coefficient of 2-KGA.
About the raising of enzyme activity, patent document 1 finds Fe3+、Mg2+And Ca2+L-sorbose dehydrogenase and L- can be effectively improved
The activity of sorbic ketone dehydrogenase, but Cu2+Then inhibit L-Sorbitone Dehydrogenase active, excessive Cu2+Cause huge in fermentation process
Bacillus (B.megaterium) self-dissolving, to influence the yield of 2-KGA, and adds Mg2+And Mn2+It then can effectively inhibit this
One phenomenon.Therefore, patent document 1 realizes raising and being properly added these metal ions in the medium and effectively controlling enzyme activity
The purpose of 2-KGA yield.
Patent document 2 also optimizes culture medium, by add reduced glutathione make thallus resist osmotic pressure and
The ability for maintaining normal redox environment in vivo promotes mixed thallus system preferably to adapt to yeasting, improves ketone 2-KLG
Bacteria biomass.Patent document 3 is then on the basis of the sequencing of size bacterium full-length genome and functional annotation, to size bacterium glycometabolism net
Network is analyzed, and finds and demonstrate a batch and can be utilized and have very well by small bacterium during mixed fungus fermentation to promote thallus raw
Long cheap carbon source, such as ethyl alcohol, acetaldehyde and acetic acid (salt), D-glucitol, PEARLITOL 25C and D-MANNOSE.
About the raising of electron transmission efficiency, patent document 4 finds constituent of the lipoic acid as coenzyme or prothetic group, leads to
The function of exercising coupling acyl group transfer and electronics transfer is crossed, is played an important role in metabolism.Therefore patent document 4 by
This trophic factors is supplemented in culture medium to improve the growth of ketone 2-KLG bacterium and acid producing ability.
Existing technical literature:
Patent document 1:CN101402988A
Patent document 2:CN102424830A
Patent document 3:CN104357529A
Patent document 4:CN102321698B
Summary of the invention
Problems to be solved by the invention
The present inventor concentrates on studies to sorbose metabolic pathway production 2-KGA, it is intended to find new effective control
The key substance of enzyme activity and electron transmission efficiency, and using the substance Optimal Medium and improve fermentation method for producing.
In sorbose metabolic pathway, sorbose is catalytically conveted to sorb ketone through sorbose dehydrogenase, and sorb ketone is again through mountain
Pears ketone dehydrogenase is catalytically conveted to 2-KGA.Pyrroloquinoline quinone (Pyrroloquinoline quinone, abbreviation PQQ) is small bacterium
The coenzyme of sorbose dehydrogenase and sorbic ketone dehydrogenase on cell membrane, in sorbose catalytic process, after sorbose dehydrogenation, sorb
The coenzyme PQQ of ketone dehydrogenase is by electron transmission to cromoci, and cromoci is again by electron transmission to cytochromes terminal oxygen
Change enzyme.In sorb ketone catalytic process, it is compound that the PQQ coenzyme of ferroheme and sorbic ketone dehydrogenase collectively forms quinone-hemoprotein
Body, after sorb ketone dehydrogenation, for PQQ by electron transmission to ferroheme, electronics is further directly passed to cytochromes end by ferroheme
Hold oxidizing ferment.Cromoci and ferroheme are the key that cell membrane electron transmission speed limit substances, and content is extremely low in cell, only
Be able to satisfy the demand of cell normal growth metabolism, electron transport rate is very limited, be not able to satisfy cell fast-growth with
And the production requirement of efficient catalytic.
The characteristics of based on above-mentioned metabolic pathway, the present invention add the mixotrophism of cromoci and ferroheme by external source
Agent increases the content of cromoci and ferroheme in mixed fermentation system, promotes cell growth metabolism, while assisting coenzyme
PQQ enhances the enzymatic efficiency of sorbic ketone dehydrogenase, to achieve the purpose that improve 2-keto-L-gulonic acid combined coefficient.
Currently, the prior art not yet seeks the mixing of coenzyme PQQ, cromoci and ferroheme during real attenuation
It supports mixed fungus fermentation metabolic alterations caused by agent and carries out systematic research, for coenzyme PQQ in mixed bacterium large scale fermentation production, thin
The preferable and more economical control strategy of the mixotrophism agent of Cytochrome C and ferroheme does not also carry out system research.
The solution to the problem
The optimization of culture medium of the invention mainly passes through the mixing of the cromoci of addition certain concentration and ferroheme composition
Nutritional agents realizes that the improvement of the fermentation method for producing of 2-KGA of the invention is mainly by using the culture medium of the optimization, one
In a little preferred embodiments, further uses the culture medium and add the mixotrophism agent or coenzyme PQQ in due course to realize.
Specifically, the present invention includes following technical solution:
[1] the fermentation culture medium of a kind of 2-keto-L-gulonic acid of, containing by cromoci and blood in the culture medium
The mixotrophism agent of red pigment composition.
[2] culture medium according to [1], wherein the content of the mixotrophism agent in the medium is
0.007~0.009g/L, preferably 0.0075~0.0085g/L.
[3] culture medium according to [1] or [2], wherein the weight percent of the mixotrophism agent forms are as follows: 25
~67% cromoci and 33~75% ferroheme.
[4] is according to the described in any item culture mediums in [1]~[3], wherein the medium pH is 6.0~8.0, is contained
(g/L): L- sorbose 80~120, corn pulp 4.0~6.0, urea 11.0~13.0, potassium dihydrogen phosphate 0.5~1.5, anhydrous sulphur
Sour magnesium 0.1~0.2, calcium carbonate 4.0~6.0, the mixotrophism agent 0.007~0.009.
[5] a kind of fermentation method for producing of 2-keto-L-gulonic acid of, wherein the method includes being seeded to seed liquor
Fermented and cultured is carried out in fermentation medium, so that 2-keto-L-gulonic acid is generated, containing by cell color in the fermentation medium
The mixotrophism agent of plain C and ferroheme composition.
[6] fermentation method for producing according to [5], wherein the content of the mixotrophism agent in the medium
For 0.007~0.009g/L, preferably 0.0075~0.0085g/L.
In some preferred embodiments of the present invention, adjusted in the fermentation medium before the seed liquor is inoculated with
Mixotrophism agent concentration is 0.007~0.009g/L, preferably 0.0075~0.0085g/L.Preliminary experiment of the present invention in early period
Middle discovery can significantly improve 2- when the initial concentration of mixotrophism agent in the fermentation medium is in 0.007~0.009g/L
The yield and combined coefficient of KGA.When mixotrophism agent concentration is less than 0.007g/L, cromoci is only able to satisfy with ferroheme
The demand of cell normal growth metabolism, and the mass production of 2-KGA needs somatic cells vigorous growth, boosts metabolism, because
This needs to improve mixotrophism agent concentration.But when mixotrophism agent concentration is higher than 0.009g/L, the cell color of excessive concentrations
Plain C and ferroheme make cell overgrowth, largely consume raw material, reduce so as to cause the yield of 2-KGA, combined coefficient reduces.
It has also been found that when mixotrophism agent concentration be 0.0075~0.0085g/L when, not only may be implemented 2-KGA yield and
The raising of combined coefficient, and can effectively shorten fermentation period.
[7] fermentation method for producing according to [5] or [6], wherein the weight percent group of the mixotrophism agent
Become: 25~67% cromoci and 33~75% ferroheme.
[8] is according to the described in any item fermentation method for producing in [5]~[7], wherein the pH of the fermentation medium is 6.0
~8.0, contain (g/L): L- sorbose 80~120, corn pulp 4.0~6.0, urea 11.0~13.0, potassium dihydrogen phosphate 0.5
~1.5, anhydrous magnesium sulfate 0.1~0.2, calcium carbonate 4.0~6.0, the mixotrophism agent 0.007~0.009.
[9] is according to the described in any item fermentation method for producing in [5]~[8], wherein when mixed in the fermentation medium
When the concentration of conjunction nutritional agents is down to 0.001~0.003g/L, the mixotrophism agent is added in supplement.
In some preferred embodiments of the present invention, when the concentration of the mixotrophism agent in the fermentation medium is down to
When 0.001~0.003g/L, the mixotrophism agent is added in supplement.It is a discovery of the invention that phase after fermentation, when in fermentation medium
The concentration of mixotrophism agent when being lower than 0.001g/L, cromoci and ferroheme be not able to satisfy somatic cells fast-growth and
The needs of metabolism, 2-KGA synthesis rate reduce, and cause fermentation period to elongate, therefore should supplement mixotrophism agent in due course.Work as mixing
When the concentration of nutritional agents is higher than 0.003g/L, cell overgrowth, culture medium Central Plains will lead to if adding mixotrophism agent
Material consumption is excessive, reduces so as to cause the yield of 2-KGA.
[10] fermentation method for producing according to [9], wherein the mixotrophism agent is added to remain described in supplement
Mixotrophism agent concentration in fermentation medium is 0.001~0.003g/L, until fermentation ends.
In some preferred embodiments of the present invention, the mixotrophism agent is added to maintain the fermentation medium in supplement
In mixotrophism agent concentration be 0.001~0.003g/L, until fermentation ends.It is a discovery of the invention that according to acid amount prison in real time is produced
Survey the results show that with fed-batch mode supplement mixotrophism agent to concentration within the scope of 0.001~0.003g/L, can obtain higher
2-KGA yield and combined coefficient.Mixotrophism agent except this range will lead to cell growth metabolism deficiency or excessive,
It is unfavorable to the production of 2-KGA.
[11] fermentation method for producing according to [9] or [10], wherein supplemented described in addition in such a way that stream adds
Mixotrophism agent.
[12] is according to the described in any item fermentation method for producing in [5]~[11], wherein during the fermented and cultured,
When online oxygen dissolving value is 20%~40%, supplement be added pyrroloquinoline quinone PQQ make its concentration 0.003 in the medium~
0.005g/L, preferably when online oxygen dissolving value is 25%~35%, supplement is added.
In some preferred embodiments of the present invention, during the fermented and cultured, when online oxygen dissolving value is 20%~
When 40%, pyrroloquinoline quinone PQQ, which is added, in supplement makes its 0.003~0.005g/L of concentration in the medium, preferably when online
Supplement is added when oxygen dissolving value is 25%~35%.It is a discovery of the invention that the oxygen dissolving value during fermented and cultured influences electron transmission effect
Fruit, final receptor of the oxygen as electron transmission on cellular respiration chain, oxygen deficiency will affect the efficiency of electron transmission, but oxygen is same
When be a kind of oxidant again, oxygen excess will affect the catalytic activity of cellular enzymes, be embodied in the fermentation stage later period, when online
Lower than 20% or when being higher than 40%, the generating rate of 2-KGA can decline oxygen dissolving value.It has also been found that online oxygen dissolving value exists
When 25%~35% range, the yield of 2-KGA can be significantly improved, effectively shortens fermentation period, and then improves the synthesis of 2-KGA
Efficiency.
[13] is according to the described in any item fermentation method for producing in [5]~[12], wherein the fermentation culture conditions are as follows: temperature
Degree is 24~34 DEG C, and pH is 6.0~8.0, and speed of agitator is 200~650rpm, and ventilatory capacity is 5~18L/min, and tank pressure is 0.02
~0.08MPa, time are 40~60 hours.
[14] is according to the described in any item fermentation method for producing in [5]~[13], wherein the method also includes fermentations to tie
Separation obtains 2-keto-L-gulonic acid in fermentation medium described in Shu Houcong.
[15] is according to the described in any item fermentation method for producing in [5]~[14], wherein the seed liquor is small bacterium and companion
The mixed culture of raw bacterium, the small bacterium are ordinary student ketone group 2-KLG bacterium (Ketogulonigenium vulgare), the companion
Raw bacterium is selected from by bacillus megaterium (Bacillus megaterium), Bacillus cercus (Bacillus cereus), withered
The group that careless bacillus, bacillus thuringiensis form.
The effect of invention
Fermentation medium of the invention can effectively improve enzyme and urge because of the cromoci containing certain concentration and ferroheme
Change efficiency and electron transmission efficiency, promotes the growth metabolism of cell, and then improve the combined coefficient of 2-KGA.In addition, of the invention
2-KGA fermentation method for producing makes cromoci and ferroheme to coenzyme by the way that cromoci, ferroheme and PQQ is applied in combination
PQQ plays good synergism, the enzymatic efficiency of sorbic ketone dehydrogenase is effectively enhanced, by these three substances in electronics
The synergistic effect in chain is transmitted, while promoting cell growth metabolism, moreover it is possible to improve the combined coefficient of 2-KGA.
Culture medium and fermentation method for producing of the invention includes following advantage:
1) after medium optimization, the reasonable addition of cromoci and ferroheme can make fermentation period shorten 9.8%, 2-KGA
Combined coefficient improve 11.6%.
2) the reasonable addition of cromoci and ferroheme can make fermentation period shorten 17.6% in conjunction with the in due course supplement of PQQ,
The combined coefficient of 2-KGA improves 23.1%.
Specific embodiment
In order to better describe the present invention, following embodiment is provided for reference.It should be noted that following embodiment is only used
In the description present invention, the invention is not limited thereto.
The source of bacterial strain used in the present invention, the preparation of culture medium, preparation, fermentation culture conditions and the production of mixed culture
The measuring method of object can obtain or implement in the following way.
(1) bacterium source
Mixed culture used in the present invention including small bacterium and concomitance bacterium is unrestricted, as long as using training of the invention
In the case where supporting base, 2-KGA can be obtained by the mixed culture.The small bacterium bag includes bacterial strain commonly used in the art,
It is preferred that ordinary student ketone group 2-KLG bacterium (Ketogulonigenium vulgare), i.e. ketone 2-KLG bacterium.The concomitance bacterium includes
Bacterial strain commonly used in the art, preferably bacillus megaterium (Bacillus megaterium), Bacillus cercus
(Bacillus cereus), bacillus subtilis (Bacillus subtilis), bacillus thuringiensis (Bacillus
Thuringiensis) etc..
It is ordinary student ketone group 2-KLG that the small bacterium of mixed culture used in the following embodiment and concomitance bacterium, which are respectively as follows: small bacterium,
Bacterium (Ketogulonigenium vulgare) (it was once named as Gluconobacter oxvdans (Gluconobacter oxydans),
The deposit number of the Gluconobacter oxvdans is CGMCC NO.1.110, purchased from China General Microbiological culture presevation management
The heart, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101)；Concomitance bacterium is huge
(deposit number is CGMCC NO.1.432 to Bacterium anthracoides (Bacillus megaterium), is purchased from China General Microbiological strain
Preservation administrative center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101).
(2) preparation of culture medium
Following nutrient media components use material commonly used in the art, and preparation method is also preparation side well known in the art
The composition of method, each culture medium is as follows:
1) solid medium (g/L): L- sorbose 60~80, corn pulp 4.0~6.0, urea 11.0~13.0, phosphoric acid
Potassium dihydrogen 0.5~1.5, anhydrous magnesium sulfate 0.1~0.2, calcium carbonate 4.0~6.0, mixotrophism agent 0.007~0.009, agar
15~20, pH6.0~8.0.
2) fluid nutrient medium (g/L): L- sorbose 80~120, corn pulp 4.0~6.0, urea 11.0~13.0, phosphoric acid
Potassium dihydrogen 0.5~1.5, anhydrous magnesium sulfate 0.1~0.2, calcium carbonate 4.0~6.0, mixotrophism agent 0.007~0.009, pH6.0
~8.0.
3) fermentation medium (g/L): L- sorbose 80~120, corn pulp 4.0~6.0, urea 11.0~13.0, phosphoric acid
Potassium dihydrogen 0.5~1.5, anhydrous magnesium sulfate 0.1~0.2, calcium carbonate 4.0~6.0, mixotrophism agent 0.007~0.009, pH6.0
~8.0.
4) conventional sterilant equipment, 115 DEG C of sterilizing 25min, the wherein independent 115 DEG C of sterilizings of sorbose sterilising conditions: are used
25min.Plate is made after solid medium sterilizing.
5) preparation of mixotrophism agent: its weight percent composition are as follows: 25~67% cromoci, 33~75%
Ferroheme, without high-temperature sterilization, only filtration sterilization in superclean bench, spare.
6) preparation of coenzyme PQQ mother liquor: PQQ being added into purified water and prepares mother liquor, and PQQ concentration is 0.010g/L, is only existed
Filtration sterilization in superclean bench, it is spare.
(3) preparation of mixed culture:
Small bacterium and concomitance bacterium are seeded on solid medium respectively, 24~36h is cultivated at 28 DEG C.Then, solid is trained
Bacteria suspension is respectively prepared in the small bacterium and concomitance bacterium supported on base, respectively takes 1mL to be respectively connected to seed culture medium, while respectively 1mL being taken to mix
Access seed culture medium, triangular flask liquid amount 25mL/250mL, 28 DEG C~30 DEG C, cultivate 18 under 180~200rpm of shaking speed
It being accessed in corresponding fermentation medium after~20h with 5% inoculum concentration, triangular flask liquid amount is 25mL/250mL, 28 DEG C~30
DEG C, 180~200rpm of shaking speed, cultivate 40~60h.Residual sugar, 2-KGA content, pH value, and microscopy bacterium are detected in fermentation process
Volume morphing.
(4) fermentation culture conditions:
Temperature is 24~34 DEG C, and pH is 6.0~8.0, and speed of agitator is 200~650rpm, and ventilatory capacity is 5~18L/min,
Tank pressure is 0.02~0.08MPa, and the time is 40~60h.
(5) detection method
1) measuring method of 2-KGA content
Measuring principle:
2-KGA reacts in strong acid media through lactone enolization, is converted into VC, using VC and iodine oxidation reduction reaction, surveys
After determining VC content, it is converted to 2-KGA content.
Measuring method: the 7mol/L sulfuric acid of 2mL is added in test tube in liquid-transfering gun pipette samples 2mL, shakes up rear boiling water bath and adds
Hot 25min (2-KGA, which lactonizes, generates VC) is washed in 250mL triangular flask with purified water through 3~4 times after taking out cooling, is added
0.5% starch solution 3mL, with 0.1mol/L iodine solution be titrated to blue 30s do not take off (iodine can aoxidize VC, after VC runs out of,
Iodine meets starch and becomes blue), as titration end-point.
Calculation method:
Wherein:
C--- iodine titration solution concentration (mol/L)
V--- sample consumption iodine titration solution volume (mL)
0.9071--VC molecular weight/2-KGA molecular weight
The 0.05mol/L iodine titer of the every 1mL of 8.806--- is equivalent to the VC of 8.806mg
A---2-KGA is converted to the molar yield of VC (in terms of 63.10%)
B--- accurately draws fermentating liquid volume (mL)
The theoretical concentration of 0.05--- iodine titer
2) measuring method of residual sugar content
Residual sugar in fermentation liquid is measured using anthrone method, specifically: take fermentation broth sample 1mL in clean volumetric flask plus pure
Change water constant volume and be diluted to certain multiple, then draw 1mL dilution in clean drying test tube, 6mL anthrone solution is added, uses whirlpool
After whirlpool oscillator shakes up, 20min is stood at room temperature；Blank control replaces fermentation broth sample to carry out aforesaid operations with purified water；Benefit
With ultraviolet specrophotometer, colorimetric surveys absorbance value at wavelength 620nm, is then scaled residual sugar content.When fermentation liquid residual sugar contains
When amount≤0.5mg/mL, as fermentation termination.
3) measuring method of online oxygen dissolving value
METTLER dissolved oxygen electrode (electrode model: InPro 6800, Mei Teletuo benefit instrument (Shanghai) limited public affairs is installed
Department), fermentation adjusts each technological parameter to preset value before starting, using two-point method (correct respectively online oxygen dissolving value 0% with it is online
Oxygen dissolving value 100%) the online dissolved oxygen parameter value of correction, technological operation is then carried out according to online oxygen dissolving value.
Embodiment 1
1) mixotrophism agent, weight percent composition are as follows: 33% cromoci, 67% ferroheme are prepared.It prepares
After the completion, spare after filtration sterilization in superclean bench.
2) it prepares fermentation medium (g/L): L- sorbose 120.0, corn pulp 5.0, urea 12.0, potassium dihydrogen phosphate 1.0,
Anhydrous magnesium sulfate 0.1, calcium carbonate 5.0, mixotrophism agent 0.008 adjust pH7.0,115 DEG C of sterilizing 25min, wherein sorbose list
Only 115 DEG C of sterilizings 25min.
3) it is inoculated in fermentation medium with the kind liquid that 20% inoculum concentration will be enlarged by culture, is carried out using 15L fermentor
It ferments (liquid amount 70%), the item of 32 DEG C of temperature, pH6.8, speed of agitator 600rpm, ventilatory capacity 15L/min, tank pressure 0.03MPa
It ferments under part, synthesizes 2-KGA.To the fermentation middle and later periods, when mixotrophism agent concentration is down to 0.002g/L, using flow feeding
Mode maintains the mixotrophism agent concentration in fermentation liquid for 0.002g/L to fermentation termination.Fermentation period is 46h, and terminal acid amount is
114.62mg/mL, residual sugar 0.32mg/mL, 2-KGA combined coefficient are 2.492mg/mLh-1。
Embodiment 2~8 and comparative example 1~3
According to the method for embodiment 1, the content of cromoci and ferroheme, mixotrophism agent in mixotrophism agent are adjusted
Initial addition concentration, mixotrophism agent stream add the parameters such as concentration, other conditions are same as Example 1, as a result such as the following table 1 institute
Show:
Table 1
It is not added with cytochromes and/or ferroheme in the culture medium of comparative example 1~3 it can be seen from 1 data of table, with it
Compare, be added to the Examples 1 to 8 of cytochromes and/or ferroheme fermentation period is short and the combined coefficient of 2-KGA is high.
Wherein, be not only added in the culture medium of Examples 1 to 5 cromoci of the content in 25~67% ranges and
Ferroheme of the content in 33~75% ranges is also maintained at the concentration for being inoculated with mixotrophism agent in primary fermentation culture medium
Within the scope of 0.007~0.009g/L, and the fermentation middle and later periods also add mixotrophism agent make its concentration maintain 0.001~
Within the scope of 0.003g/L, thus in the entire fermentation process of Examples 1 to 5 cromoci and ferroheme supply it is abundant, advantageously
The growth metabolism for promoting cell realizes and obtains higher 2-KGA yield in the fermentation period more shorter than comparative example 1~3
And combined coefficient.Wherein 1 effect of embodiment is best, and compared with the comparative example 1 for being not added with mixotrophism agent, fermentation period can shorten
9.8%, 2-KGA combined coefficient improve 11.6%.
But when the content of cromoci in mixotrophism agent is not in 25~67% ranges and content of hemachrome does not exist
When in 33~75% ranges (comparative example 1~3), then it is lower will to there is long fermentation period, the yield of product 2-KGA and combined coefficient
Unfavorable result.In addition, when the concentration of mixotrophism agent in inoculation primary fermentation culture medium is not within the scope of 0.007~0.009g/L
(embodiment 8), or fail to add mixotrophism agent in due course in the fermentation middle and later periods to maintain its concentration in 0.001~0.003g/L
In range (embodiment 6~7), then compared with Examples 1 to 5, the combined coefficient of fermentation period length or product 2-KGA are slightly lower,
But still shorter than the fermentation period of comparative example 1~3 and 2-KGA combined coefficient is high.
Therefore, cromoci and crucial speed limit substance of the ferroheme as cell membrane electron transmission, appropriate adjustment its first
Content in beginning culture medium and fermentation middle and later periods can promote the growth metabolism of cell, improve enzyme activity (Examples 1 to 5).But
The cromoci and ferroheme of excessive concentrations can make cell overgrowth, excessively consume raw material, and the yield of 2-KGA is caused to reduce,
Combined coefficient reduces (embodiment 7~8).
Embodiment 9
1) mixotrophism agent, weight percent composition are as follows: 33% cromoci, 67% ferroheme are prepared.Preparation
Coenzyme PQQ mother liquor, wherein coenzyme PQQ concentration is 0.10g/L.It is spare respectively at filtration sterilization in superclean bench.
2) it prepares fermentation medium (g/L): L- sorbose 120.0, corn pulp 5.0, urea 12.0, potassium dihydrogen phosphate 1.0,
Anhydrous magnesium sulfate 0.1, calcium carbonate 5.0, mixotrophism agent 0.008 adjust pH7.0,115 DEG C of sterilizing 25min, wherein sorbose list
Only 115 DEG C of sterilizings 25min.
3) it is inoculated in the fermentation medium for being added to mixotrophism agent with the kind liquid that 20% inoculum concentration will be enlarged by culture,
It is fermented (liquid amount 70%) using 15L fermentor, 32 DEG C of temperature, pH6.8, speed of agitator 600rpm, ventilatory capacity 12L/
It ferments under conditions of min, tank pressure 0.03MPa, when mixotrophism agent concentration drops to 0.002g/L, is tieed up in such a way that stream adds
Mixotrophism agent concentration 0.002g/L is held in fermentation liquid to fermentation termination.When oxygen dissolving value online in fermentation technology process is down to 30%
When add PQQ, coenzyme PQQ is added by the way of flow feeding, PQQ concentration is made in fermentation liquid to maintain 0.004g/L to fermenting
Terminal.Fermentation period is 42h, and terminal acid amount is 115.02mg/mL, and residual sugar 0.37mg/mL, 2-KGA combined coefficient is
2.748mg/mL·h-1。
Embodiment 10~13 and reference example 1~3
According to the method for embodiment 9, the parameters such as the PQQ concentration after adding, stream added-time online oxygen dissolving value, other conditions are flowed in adjustment
It is same as Example 9, as a result as shown in table 2 below:
Table 2
It can be seen from the data in table 2 compared with not flowing the reference example 1 for adding PQQ, since embodiment 9~13 is being fermented
Stream plus PQQ when the online oxygen dissolving value of culture is 20%~40%, and make its 0.003~0.005g/ of concentration in the medium
L, therefore fermentation period is effectively shortened, improve 2-KGA yield and combined coefficient.Wherein the effect of embodiment 9 is best, with ginseng
It examines example 1 to compare, cycle time 8.7%, 2-KGA combined coefficient improves 10.3%, adds with neither adding mixotrophism agent nor flowing
The comparative example 1 of PQQ is compared, cycle time 17.6%, and 2-KGA combined coefficient improves 23.1%.
Corresponding thereto, reference example 2~3 is not due to adding PQQ in dissolved oxygen opportunity appropriate stream or flowing the PQQ concentration mistake added
Height, therefore cause longer fermentation period, 2-KGA yield and combined coefficient lower.
Result above is analyzed, inventors believe that, PQQ is as coenzyme, with sorbose dehydrogenase and sorbic ketone dehydrogenase knot
The enzymatic efficiency that the two can be improved is closed, so when cell enters stationary phase, sorbose dehydrogenase and sorbic ketone dehydrogenase
Concentration, which reaches, adds suitable PQQ after saturation state again and can effectively improve the catalytic efficiency of enzyme, but PQQ pairs of excessive concentrations
The growth metabolism of cell has certain toxic action, is unfavorable for the synthesis of 2-KGA.
Industrial availability
Cromoci, ferroheme and coenzyme PQQ are the related enzyme activity and electron transmission effect improved in sorbose metabolic pathway
The key factor of rate can effectively facilitate the growth metabolism of cell, improve the yield and combined coefficient of 2-KGA.Use this hair
Culture medium after bright optimization, and the mixotrophism agent of cromoci and ferroheme composition is added in due course, fermentation period can be made to contract
The combined coefficient of short 9.8%, 2-KGA improves 11.6%；It is further improved fermentation method for producing on this basis, i.e., in fermentation
Moment adds PQQ in due course, and the combined coefficient that fermentation period can be made to shorten 17.6%, 2-KGA improves 23.1%.Therefore, originally
The culture medium and fermentation process of invention are of great significance for industrialization large-scale production 2-KGA.
Claims (15)
1. a kind of fermentation culture medium of 2-keto-L-gulonic acid, containing by cromoci and ferroheme group in the culture medium
At mixotrophism agent.
2. culture medium according to claim 1, wherein the content of the mixotrophism agent in the medium is
0.007~0.009g/L, preferably 0.0075~0.0085g/L.
3. culture medium according to claim 1 or 2, wherein the weight percent of the mixotrophism agent forms are as follows: 25~
67% cromoci and 33~75% ferroheme.
4. described in any item culture mediums according to claim 1~3, wherein the medium pH is 6.0~8.0, is contained
(g/L): L- sorbose 80~120, corn pulp 4.0~6.0, urea 11.0~13.0, potassium dihydrogen phosphate 0.5~1.5, anhydrous sulphur
Sour magnesium 0.1~0.2, calcium carbonate 4.0~6.0, the mixotrophism agent 0.007~0.009.
5. a kind of fermentation method for producing of 2-keto-L-gulonic acid, wherein the method includes seed liquor is seeded to fermentation training
Support in base and carry out fermented and cultured, to generate 2-keto-L-gulonic acid, in the fermentation medium containing by cromoci and
The mixotrophism agent of ferroheme composition.
6. fermentation method for producing according to claim 5, wherein the content of the mixotrophism agent in the medium
For 0.007~0.009g/L, preferably 0.0075~0.0085g/L.
7. fermentation method for producing according to claim 5 or 6, wherein the weight percent of the mixotrophism agent forms
Are as follows: 25~67% cromoci and 33~75% ferroheme.
8. according to the described in any item fermentation method for producing of claim 5~7, wherein the pH of the fermentation medium be 6.0~
8.0, contain (g/L): L- sorbose 80~120, corn pulp 4.0~6.0, urea 11.0~13.0, potassium dihydrogen phosphate 0.5~
1.5, anhydrous magnesium sulfate 0.1~0.2, calcium carbonate 4.0~6.0, the mixotrophism agent 0.007~0.009.
9. according to the described in any item fermentation method for producing of claim 5~8, wherein the mixing in the fermentation medium
When the concentration of nutritional agents is down to 0.001~0.003g/L, the mixotrophism agent is added in supplement.
10. fermentation method for producing according to claim 9, wherein the mixotrophism agent is added to remain described in supplement
Mixotrophism agent concentration in fermentation medium is 0.001~0.003g/L, until fermentation ends.
11. fermentation method for producing according to claim 9 or 10, wherein supplemented in such a way that stream adds and described mix is added
Close nutritional agents.
12. according to the described in any item fermentation method for producing of claim 5~11, wherein during the fermented and cultured, when
When online oxygen dissolving value is 20%~40%, supplement be added pyrroloquinoline quinone make its concentration 0.003 in the medium~
0.005g/L, preferably when online oxygen dissolving value is 25%~35%, supplement is added.
13. according to the described in any item fermentation method for producing of claim 5~12, wherein the fermentation culture conditions are as follows: temperature
Be 24~34 DEG C, pH is 6.0~8.0, and speed of agitator is 200~650rpm, and ventilatory capacity is 5~18L/min, tank pressure for 0.02~
0.08MPa, time are 40~60 hours.
14. according to the described in any item fermentation method for producing of claim 5~13, wherein the method also includes fermentation ends
Separation obtains 2-keto-L-gulonic acid from the fermentation medium afterwards.
15. according to the described in any item fermentation method for producing of claim 5~14, wherein the seed liquor is small bacterium and association
The mixed culture of bacterium, the small bacterium are ordinary student ketone group 2-KLG bacterium (Ketogulonigenium vulgare), the association
Bacterium is selected from by bacillus megaterium (Bacillus megaterium), Bacillus cercus (Bacillus cereus), withered grass
The group that bacillus (Bacillus subtilis), bacillus thuringiensis (Bacillus thuringiensis) form.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1685050A (en) * | 2002-09-27 | 2005-10-19 | 帝斯曼知识产权资产管理有限公司 | Process for producing vitamin C |
| CN102321698A (en) * | 2011-08-16 | 2012-01-18 | 中国人民解放军军事医学科学院生物工程研究所 | Method for promoting growth and acid production of Ketogulonigenium vulgare |
| 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
- 2018-12-28 CN CN201811626312.8A patent/CN109593813B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1685050A (en) * | 2002-09-27 | 2005-10-19 | 帝斯曼知识产权资产管理有限公司 | Process for producing vitamin C |
| CN102321698A (en) * | 2011-08-16 | 2012-01-18 | 中国人民解放军军事医学科学院生物工程研究所 | Method for promoting growth and acid production of Ketogulonigenium vulgare |
| 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 (2)
| Title |
|---|
| JIN DU等: "Combinational expression of sorbose/sorbosone dehydrogenases and cofactor pyrroloquinoline quinone increases 2-keto-L-gulonic acid production in Ketogulonigenium vulgare-Bacillus cereus consortium", 《METABOLIC ENGINEERING》 * |
| 李野等: "酮古龙酸菌与呼吸链偶联的2-KGA代谢途径研究进展", 《微生物学报》 * |
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