CN105255959B - A kind of feed process promoting Rifamycin Sodium fermentation combined coefficient - Google Patents
A kind of feed process promoting Rifamycin Sodium fermentation combined coefficient Download PDFInfo
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- CN105255959B CN105255959B CN201510682696.5A CN201510682696A CN105255959B CN 105255959 B CN105255959 B CN 105255959B CN 201510682696 A CN201510682696 A CN 201510682696A CN 105255959 B CN105255959 B CN 105255959B
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Abstract
The invention discloses a kind of feed process of promotion Rifamycin Sodium fermentation combined coefficient.The three grade fermemtation culture Amycolatopsis mediterranei period be 48-72 h when, start into fermentation culture medium it is sterile stream plus acetaldehyde solution, until before fermentation ends 10 h stop stream plus, make the final concentration of 1.5-3.0 mmol/L of acetaldehyde.Rifamycin Sodium potency averagely improves 10.1% at the end of culture.The new method has the characteristics that easy to operate, Rifamycin Sodium effect of increasing production is stablized.
Description
Technical field
The present invention relates to a kind of feed process of promotion Rifamycin Sodium fermentation combined coefficient.The invention belongs to bio-pharmaceuticals
Field of fermentation engineering regulates and controls the migration of oxidation direction and efficiency, improvement carbon, nitrogen of NADH intracellular more particularly to a kind of fermentation process
Metabolic fluxes distribute, promote the generation of the core azo-cycle precursor 3- amino -5- hydroxybenzoic acid of Rifamycin Sodium, improve rifamycin
The method of SV combined coefficient.
Background technique
As large antibiotic bulk pharmaceutical chemicals of tuberculotherapy, rifamycin fermentation combined coefficient is improved always by production
Enterprise and research unit's concern.Currently, domestic more about the report for improving rifamycin fermentation titer, comprising: production strain
Molecular biology information characteristics, fermentation medium optimization etc., such as documents 1: shaking flask level add soya-bean oil and third
Rifamycin Sodium potency 12.3% (food and fermentation industries, Shang Chunliang, 2011,37 (5): 56-60) can be improved in propylhomoserin；Comparison text
Offer 2: adding soya-bean oil and ethanedioic acid in 50 tons big tank level can make Rifamycin Sodium fermentation 1,000,000,000 improve 5.4% (patent of invention
ZL201210176175.9)；Documents 3: addition alanine, soya-bean oil and fosfomycin sodium/fosfomycin trometamol can make Li Fu
Mycin SV potency averagely improves 17.0% (application number of invention patent 201210176177.8).Documents 4: in 10L fermentor
To Rifamycin Sodium potency and there is base-material addition zinc ion (final concentration 0.75mg/L) during culture Amycolatopsis mediterranei
Machine acid, which has been synthetically produced, significantly affects (China's brewing, Deng Pengfei, 2013,32 (8): 84-87).But documents 1 pertain only to
There are reciprocations for soya-bean oil and alanine, and under determining optimization concentration, Rifamycin Sodium potency improves 12.3%, and to production
Glycolytic pathway carbon metablism, organic acid combined coefficient and the influence of potency synthesis of strain have carried out preliminary analysis, not
The influence that the migration of oxidation direction and efficiency for relating to NADH in fermentation process synthesize Rifamycin Sodium；Documents 2 are related to
The double action of soya-bean oil and ethanedioic acid adjusts the committed step in tricarboxylic acid cycle --- the work of the reaction of succinate dehydrogenase catalysis
Property, passing through and promote Rifamycin Sodium precursor --- the anabolism efficiency of methylmalonyl CoA improves the synthesis of Rifamycin Sodium
Efficiency, the influence that also the oxidation direction migration without reference to NADH in fermentation process and efficiency synthesize Rifamycin Sodium；Comparison
Document 3 only tentatively illustrates that three has the function of adjusting Rifamycin Sodium fermentation, and the document was also without reference to fermenting
The influence that the oxidation direction migration of NADH and efficiency synthesize Rifamycin Sodium in journey；Documents 4 pass through analysis extracellular portion
The changing rule of organic acid, amino acid, report zinc ion are made by inhibiting alanine dehydrogenase activity and causing acetone acid accumulation
The lysine as derived from oxaloacetic acid and the glutamic acid synthetic quantity as derived from α-ketoglutaric acid increase, and promote Rifamycin Sodium
Synthesis.But documents 4 do not relate to the oxidation direction migration of NADH in fermentation process and efficiency closes Rifamycin Sodium
At influence.
Redox reaction almost participates in the links of growth of microbial cells and metabolism, in aerobic respiration condition
Under, NADH is oxidized to NAD through electron transport chain+, and a large amount of ATP are generated, the synthesis for cell activities and product provides energy
Amount.But electron transmission is restricted under fermentation conditions, and carbon metablism approach is caused to change.During cell growth metabolism
Glycolytic pathway (EMP) and tricarboxylic acid cycle (TCA) can largely generate NADH, if under fermentation conditions cell cannot well by
NADH is aoxidized through electron transport chain or some dehydrogenase catalyzed reactions (such as: alanine dehydrogenase), just will appear NADH/NAD+
Proportional imbalance.This necessarily will affect the physiological metabolism of cell and the accumulation of secondary metabolites.Azo-cycle precursor (the 3- of Rifamycin Sodium
Amino -5- hydroxybenzoic acid) vigor of combined coefficient and glutamine synthelase (GS) is positively correlated closely and by alanine (Ala)
Inhibit, and for cell by alanine dehydrogenase oxidation NADH generation Ala, which can inhibit GS living under fermentation condition
The combined coefficient of property and 3- amino -5- hydroxybenzoic acid.Therefore, if can change NADH under fermentation condition oxidation direction and
Efficiency, it may be assumed that reduce NADH through alanine dehydrogenase catalytic route carbon flow, then can make NADH/NAD under fermentation condition+It is in
Imbalance state, and the accumulation of Ala can be reduced, so that GS activity is maintained higher level, to promote 3- amino -5- hydroxybenzoic acid
With the combined coefficient of Rifamycin Sodium.Therefore, select the suitable hydrogen acceptor of cell that may realize above-mentioned purpose under fermentation condition.
In early-stage study and production, we determined that the concentration of ethyl alcohol is quite high in fermentation process fermentation liquid, and Mediterranean
Amycolatosis cell is NADH and synthesizing alcohol to be aoxidized by alcohol dehydrogenase, and hydrogen acceptor is acetaldehyde molecule.Having
Machine chemical industry, acetaldehyde are common two carbon reagents, can also construct heterocycle ring system；It can be used for deploying fruit essence in food service industry,
It can also be used for brewed wine essence, concentration is about 3.9-270mg/kg in flavoured food product.But not about external source acetaldehyde point
Son acted on as the metabolic regulation factor Amycolatopsis mediterranei new function report, it may be assumed that make the oxidative pathway of NADH by
The reaction of alanine dehydrogenase catalysis is migrated to alcohol dehydrogenase enzymatic direction, and is increased by promoting the activity of alcohol dehydrogenase
The oxidation of strong NADH, the synthesis for reducing alanine adjust the oxidation direction of NADH and efficiency in fermentation process, improve rifamycin
The report of the new function of SV azo-cycle precursor (3- amino -5- hydroxybenzoic acid) combined coefficient, promotion fermentation yield of rifamycin SV.
Summary of the invention
The purpose of the present invention is to provide a kind of new feed process of promotion Rifamycin Sodium fermentation combined coefficient, essence
Property is not come acetaldehyde as two carbon reagent of organic chemical industry, the food service industry preparing essence on conventional meaning using but by second
Aldehyde molecule promotes the oxidation migration and efficiency, enhancing Rifamycin Sodium azo-cycle of NADH in fermentation process as the metabolic regulation factor
The combined coefficient of precursor 3- amino -5- hydroxybenzoic acid, the fermentation yield for improving Rifamycin Sodium.
When the three grade fermemtation 48-72h of Amycolatopsis mediterranei, before starting sterile stream plus acetaldehyde solution to fermentation ends
10h makes its final concentration of 1.5-3.0mmol/L, and Rifamycin Sodium potency averagely improves 10.1% at the end of culture.
The invention has the characteristics that material is easily purchased, operating procedure is simple, controllability is high, is suitable for industrial fermentation and produces.
A kind of feed process promoting Rifamycin Sodium fermentation combined coefficient proposed by the present invention, comprising the following steps:
(1) preparation of culture medium
1. seed culture medium
Glucose 1.5%, soybean cake powder 1.0%, peptone 1.0%, potassium nitrate 0.05%, calcium carbonate 0.2% are soluble
Starch 1.5%, pH 7.0.It is spare through 0.1MPa steam sterilization 30min；
2. fermentation medium
Glucose 6-10%, fish meal 0.3-0.55%, soybean cake powder 0.5-0.65%, peptone 0.4-0.7%, potassium nitrate
0.6-0.8%, calcium carbonate 0.5%, potassium dihydrogen phosphate 0.02%, pH are natural.It is spare through 0.1MPa steam sterilization 20min.
(2) actication of culture
Amycolatopsis mediterranei (A.mediterranei) strain for hiding of going bail for accesses seed culture medium, trains on shaking table
48h is supported, condition of culture is 28 DEG C, 220r/min.
(3) strain expands culture and closes the collection of bottle seed cell
Above-mentioned strain is connected in fermentation shake flask culture medium, liquid amount is 50-100mL/250mL triangular flask, and inoculum concentration is
3%-5%, in 27 DEG C -28 DEG C, 150-200r/min shaken cultivation 46-50h, shake-flask seed culture medium is closed in rear sterile working
Bottle.(4) secondary seed culture and three grade fermemtation
Conjunction bottle seed flame is protected and accesses secondary seed tank, 28 DEG C of cultivation temperature, revolving speed 160-200r/min, air
Flow control is 0.8vvm, and tank is voltage-controlled to be made as 0.03-0.04MPa, cultivates after 48h culture transferring to three grade fermemtation tank.
(5) three grade fermemtation tank controls
28 DEG C of cultivation temperature, revolving speed 0-48h be 200r/min, 48-72h 220r/min, 72-96h 230r/min,
96h is 240r/min to tank is put.Air flow control: 0-48h 0.4vvm, 48-72h 0.6vvm, 72-96h 0.8vvm,
96h is 1vvm to tank is put.The voltage-controlled system of tank: 0-48h 0.02MPa, 48-72h 0.03MPa, 72-96h 0.04MPa, 96h are extremely
Putting tank is 0.05MPa.
(6) when three grade fermemtation cultivation cycle is 48-72h, starts stream plus fill into 10h before acetaldehyde solution to fermentation ends, make
Its final concentration of 1.5-3.0mmol/L, other control techniques are constant.
Compared with prior art the invention has the advantages that and significant progress: operated by the technical program, will
The external source acetaldehyde for commonly using two carbon reagents and food service industry deodorant tune as organic chemical industry's industry is acted on as the metabolic regulation factor
Amycolatopsis mediterranei, have adjusted fermentation process NADH oxidation migration and efficiency, make ferment middle and later periods lactic acid, succinic acid,
The concentration reduction of malic acid, alanine, aminoglutaric acid concentration greatly improve, and have more efficiently facilitated 3- amino -5- hydroxybenzoic acid
Synthesis, Rifamycin Sodium potency averagely improves 10.1% at the end of culture.This be it is most apparent different from the prior art, have exceeded
Expection to Rifamycin Sodium facilitation effect produces unexpected technical effect.
Detailed description of the invention
Fig. 1 be stream plus acetaldehyde to biomass (zero, control group；●: addition group) and Rifamycin Sodium (, control group；■,
Addition group) synthesis influence diagram, data are the average value of parallel test three times.
As shown in Figure 1: acetaldehyde is added in constant speed flowing since thallus enters logarithmic growth phase (48h), keeps its final concentration of
1.5mmol/L, fermentation later period promote the growth of biomass, are higher than control group 16.5%-83.7%, and potency also obtains accordingly
It improves, in 121h, addition group potency ratio control group improves 23.3%, when fermentation ends, and the control of Rifamycin Sodium potency ratio mentions
It is high by 10.1%.
Fig. 2 be stream plus acetaldehyde to lactic acid (zero, control group；●: addition group) and succinic acid (, control group；■, addition group)
The influence diagram of synthesis, data are average value ± standard deviation.
As shown in Figure 2: with the progress of fermentation, the concentration of lactic acid, succinic acid in stream plus acetaldehyde experimental group than control group
Low, especially in 96h, the amount of lactic acid is 35mg/L in stream plus group, and the concentration of succinic acid is 36mg/L, respectively only control group
88.5%, and 41.9%.In microbial body, glycolysis generate pyruvic acid while also generate equivalent NADH, when dissolved oxygen not
When sufficient, the electronics that oxidation operation is released can be directly passed to substrate itself by fermenting by thallus, generate various differences
Metabolite, such as lactic acid and succinate fermentative.We flowed in thallus fermentation process plus acetaldehyde after, production quantity reduce,
Carbon metabolism flow is reduced to organic acid compound direction after illustrating stream plus acetaldehyde, has turned to alcohols compound direction instead.
Fig. 3 be stream plus acetaldehyde to malic acid (zero, control group；●: addition group) and alanine (, control group；■ adds
Add group) synthesis influence diagram, data be average value ± standard deviation.
As shown in Figure 3: with the progress of fermentation, the malic acid in stream plus acetaldehyde experimental group is lower than the concentration of control group,
Especially in 96h, stream adds the concentration of group malic acid to be 138mg/L, only control group 55.4%.In microbial body, glycolysis
The NADH that equivalent is also generated while generating pyruvic acid, when dissolved oxygen is inadequate, thallus can be by fermenting oxidation operation
The electronics of releasing is directly passed to substrate itself, generates a variety of different metabolites, such as: malic acid acid fermentation.We
It is flowed in thallus fermentation process after adding acetaldehyde, production quantity is reduced, and reduces carbon metabolism flow to organic acid conjunction after illustrating stream plus acetaldehyde
At direction, alcohols compound direction has been turned to instead.In addition, alanine is denseer than control group in stream plus group with the progress of fermentation
Low, 112h is spent, stream plus group alanine concentration are 5.6mg/L, only the 26.3% of control group.Illustrate stream plus acetaldehyde as NADH's
Exogenous electron receptor, is reduced into ethyl alcohol, so that transformation efficiency of the pyruvic acid to alanine is reduced, and alanine and glutamy
Inversely, when alanine concentration reduces, GS enzyme activity rises the activity of amine synzyme (GS), and the amount of glutamic acid is also corresponding
It increases, so that more glutamic acid are converted to glutamine under the action of GS enzyme, the amide nitrogen of glutamine is 3- ammonia again
Base -5- hydroxybenzoic acid (AHBA) direct donor, thereby promoting the synthesis of Rifamycin Sodium.
Fig. 4 be stream plus acetaldehyde to glutamic acid (zero, control group；●: addition group) and phenylalanine (, control group；■ adds
Add group) influence diagram of synthesis, (data be average value ± standard deviation).
As shown in Figure 4: with the progress of fermentation, stream plus group Glutamic Acid are higher than the concentration of control group, 112h, stream plus group paddy
Propylhomoserin concentration is 2.3g/L, improves 29% than control；And flow plus the phenylalanine of group the phase is lower than control after fermentation, when 120h,
The concentration of stream plus group phenylalanine is 0.238mg/L, 17.1% only compareed.From result above, we analyze stream plus acetaldehyde is made
For the exogenous electron receptor of NADH, ethyl alcohol is reduced by it, so that transformation efficiency of the pyruvic acid to alanine is reduced, and the third ammonia
Inversely, when alanine concentration reduces, GS enzyme activity rises the activity of acid and glutamine synthelase (GS), and glutamic acid
Amount also accordingly increases, so that more glutamic acid are converted to glutamine, the amide of glutamine under the action of GS enzyme
Nitrogen is 3- amino -5- hydroxybenzoic acid (AHBA) direct donor again, and thereby promoting the synthesis of Rifamycin Sodium.
Specific embodiment
Embodiment 1
1, the preparation of culture medium
(1) seed culture medium
Glucose 1.5g, soybean cake powder 1.0g, peptone 1.0g, potassium nitrate 0.05g, calcium carbonate 0.2g, soluble starch
1.5g is settled to 100mL, pH 7.0.It is spare through 0.1MPa autoclaving 30min；
(2) fermentation medium
Glucose 6-10g, fish meal 0.3-0.55g, soybean cake powder 0.5-0.65g, peptone 0.4-0.7g potassium nitrate 0.6-
0.8g, calcium carbonate 0.5g, potassium dihydrogen phosphate 0.02g are settled to 100mL, and pH is natural.Through 0.1MPa autoclaving 30min
It is spare；
2, actication of culture
Amycolatopsis mediterranei (A.mediterranei) strain for hiding of going bail for accesses seed culture medium, trains on shaking table
48h is supported, condition of culture is 28 DEG C, 220r/min；
3, strain expands culture and closes the collection of bottle cell
Above-mentioned strain is connected in fermentation shake flask culture medium, liquid amount be 50mL/250mL triangular flask, inoculum concentration 5%,
In 27 DEG C, 150r/min shaken cultivation 46h, then shake-flask seed culture medium is closed bottle by sterile working；
4, secondary seed culture and three grade fermemtation
Conjunction bottle seed flame is protected and accesses secondary seed tank, 28 DEG C of cultivation temperature, revolving speed 160r/min, air mass flow
Control is 0.8vvm, and tank is voltage-controlled to be made as 0.03MPa, and 48h sterile working fills into three grade fermemtation tank；
5, three grade fermemtation tank controls
28 DEG C of cultivation temperature, revolving speed 0-48h be 200r/min, 48-72h 220r/min, 72-96h 230r/min,
96h is 240r/min to tank is put.Air flow control: 0-48h 0.4vvm, 48-72h 0.6vvm, 72-96h 0.8vvm,
96h is 1vvm to tank is put.The voltage-controlled system of tank: 0-48h 0.02MPa, 48-72h 0.03MPa, 72-96h 0.04MPa, 96h
It is 0.05MPa to tank is put.
6, regulatory factor is added
Commercially available acetaldehyde sterile working is mixed with sterile water, when fermentation period 48h, is started sterile working and is added acetaldehyde solution stream
Make the final concentration of 1.5mmol/L of acetaldehyde to three grade fermemtation tank (100L) until 10h stopping stream adding before fermentation ends.
Embodiment 2
1, the preparation of culture medium: with embodiment 1.
2, actication of culture: with embodiment 1.
3, strain expands culture and closes the collection of bottle cell: with embodiment 1.
4, secondary seed culture and three grade fermemtation: with embodiment 1.
5, three grade fermemtation tank controls: with embodiment 1.
6, regulatory factor is added
Commercially available acetaldehyde sterile working is mixed with sterile water, when fermentation period is 60h, starts sterile working for acetaldehyde solution stream
Three grade fermemtation tank (100L) is added to, until 10h stopping stream adding before fermentation ends, makes the final concentration of 2.0mmol/L of acetaldehyde.
Embodiment 3
1, the preparation of culture medium: with embodiment 1.
2, actication of culture: with embodiment 1.
3, strain expands culture and closes the collection of bottle cell: with embodiment 1.
4, secondary seed culture and three grade fermemtation: with embodiment 1.
5, three grade fermemtation tank controls: with embodiment 1.
6, regulatory factor is added
Commercially available acetaldehyde sterile working is mixed with sterile water, when fermentation period is 72h, starts sterile working for acetaldehyde solution stream
Three grade fermemtation tank (100L) is added to, until 10h stopping stream adding before fermentation ends, makes the final concentration of 3.0mmol/L of acetaldehyde.
Claims (1)
1. it is a kind of promote Rifamycin Sodium fermentation combined coefficient feed process, it is characterised in that: Amycolatopsis mediterranei into
Row three grade fermemtation culture starts into culture sterile stream plus acetaldehyde solution to fermenting when the three grade fermemtation period is in 48-72 h
Terminate preceding 10 h, makes the final concentration of 1.5-3.0 mmol/L of acetaldehyde.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660598A (en) * | 2012-05-31 | 2012-09-12 | 河南省南街村（集团）有限公司 | Method for improving fermentation yield of rifamycin SV |
| CN102703541A (en) * | 2012-05-31 | 2012-10-03 | 河南省南街村（集团）有限公司 | Feeding method for improving rifamycin SV fermentation yield |
| CN104357529A (en) * | 2014-10-15 | 2015-02-18 | 沈阳药科大学 | Method for improving production capacity of 2-KGA (2-keto-L-gulonic acid) through enhancement of Ketogulonogeniumvulgarum carbon metabolism level |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660598A (en) * | 2012-05-31 | 2012-09-12 | 河南省南街村（集团）有限公司 | Method for improving fermentation yield of rifamycin SV |
| CN102703541A (en) * | 2012-05-31 | 2012-10-03 | 河南省南街村（集团）有限公司 | Feeding method for improving rifamycin SV fermentation yield |
| CN104357529A (en) * | 2014-10-15 | 2015-02-18 | 沈阳药科大学 | Method for improving production capacity of 2-KGA (2-keto-L-gulonic acid) through enhancement of Ketogulonogeniumvulgarum carbon metabolism level |
Non-Patent Citations (1)
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| 利福霉素B产生菌的推理选育及发酵工艺优化;孟根水;《中国优秀硕士学位论文全文数据库工程科技I辑》;20021215;B016-244 |
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