CN105255959A - Feed supplementing method capable of promoting fermentation synthesis efficiency of rifamycin SV - Google Patents
Feed supplementing method capable of promoting fermentation synthesis efficiency of rifamycin SV Download PDFInfo
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- CN105255959A CN105255959A CN201510682696.5A CN201510682696A CN105255959A CN 105255959 A CN105255959 A CN 105255959A CN 201510682696 A CN201510682696 A CN 201510682696A CN 105255959 A CN105255959 A CN 105255959A
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- acetaldehyde
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Abstract
The invention discloses a feed supplementing method capable of promoting the fermentation synthesis efficiency of rifamycin SV. An acetaldehyde solution is fed aseptically in a flowing manner into a fermentation cultivation material when the period of three-stage fermentation cultivation of Amycolatopsis mediterranei is 48-72 h, flowing feeding is stopped 10 h before fermentation ending, and the final concentration of acetaldehyde is enabled to be 1.5-3.0 mmol/L. After the cultivation is ended, the titer of the rifamycin SV is increased averagely by 10.1%. The novel method has the characteristics of simple operation, stable rifamycin SV yield increasing effect and the like.
Description
Technical field
The present invention relates to a kind of feed process promoting Rifamycin Sodium fermentation combined coefficient.The invention belongs to bio-pharmaceuticals field of fermentation engineering, particularly relate to the oxidation direction migration of NADH in a kind of fermenting process regulation and control born of the same parents and efficiency, carbon and nitrogen metabolism flow assignment of improving, promote the generation of core azo-cycle precursor 3-amino-5-hydroxy-benzoic acid of Rifamycin Sodium, improve the method for Rifamycin Sodium combined coefficient.
Background technology
As large microbiotic bulk drug of tuberculotherapy, improve rifomycin fermentation combined coefficient and enjoy manufacturing enterprise and research unit to pay close attention to always.At present, the domestic report about improving rifomycin fermentation titer is more, comprise: the aspect such as molecular biology information characteristics, fermention medium optimization producing bacterial classification, as documents 1: add soya-bean oil and L-Ala in shaking flask level and can improve Rifamycin Sodium and to tire 12.3% (food and fermentation industries, Shang Chunliang, 2011,37 (5): 56-60); Documents 2: Rifamycin Sodium fermentation 1,000,000,000 can be made to improve 5.4% (patent of invention ZL201210176175.9) at 50 tons of large tank levels interpolation soya-bean oil and oxalic acid; Documents 3: add L-Ala, soya-bean oil and fosfomycin sodium/fosfomycin trometamol and Rifamycin Sodium can be made to tire average improve 17.0% (application for a patent for invention number 201210176177.8).Documents 4: base-material adds zine ion (final concentration 0.75mg/L) and to tire on Rifamycin Sodium and organic acid synthesis creates remarkably influenced (China brewages in 10L fermentor cultivation Amycolatopsis mediterranei process, Deng Pengfei, 2013,32 (8): 84-87).But, documents 1 only relates to soya-bean oil and L-Ala exists interaction, under the optimization concentration determined, Rifamycin Sodium is tired raising 12.3%, and preliminary analysis has been carried out on glycolytic pathway carbon metablism, the organic acid combined coefficient and the impact of synthesizing of tiring of producing bacterial classification, do not relate to the impact that oxidation direction is moved and efficiency is synthesized Rifamycin Sodium of NADH in fermenting process; Documents 2 relates to committed step---the activity of the reaction of succinodehydrogenase catalysis in the dual function adjustment tricarboxylic acid cycle of soya-bean oil and oxalic acid, by promoting Rifamycin Sodium precursor---the anabolism efficiency of methylmalonyl CoA improves the combined coefficient of Rifamycin Sodium, does not also relate to the impact that oxidation direction is moved and efficiency is synthesized Rifamycin Sodium of NADH in fermenting process; Documents 3 only tentatively describes three and has the effect regulating Rifamycin Sodium fermentation, and the document does not relate to the impact that oxidation direction is moved and efficiency is synthesized Rifamycin Sodium of NADH in fermenting process yet; Documents 4 is by analyzing born of the same parents outer part organic acid, amino acid whose Changing Pattern, report zine ion is by suppressing alanine dehydrogenase activity and causing pyruvic acid to accumulate, make the Methionin derived by oxaloacetic acid and the L-glutamic acid resultant quantity increase derived by α-ketoglutaric acid, facilitate the synthesis of Rifamycin Sodium.But documents 4 does not relate to the impact that oxidation direction is moved and efficiency is synthesized Rifamycin Sodium of NADH in fermenting process.
Redox reaction almost participates in the links of growth of microbial cells and metabolism, and under aerobic repiration condition, NADH is oxidized to NAD through electron transport chain
+, and produce a large amount of ATP, for the synthesis of cell activities and product provides energy.But under fermentation conditions electron transmission is restricted, and carbon metablism approach is caused to change.In Growth of Cells metabolic process, glycolytic pathway (EMP) and tricarboxylic acid cycle (TCA) can produce NADH in a large number, if under fermentation conditions NADH can not well be oxidized through electron transport chain or some dehydrogenase catalyzed reactions (as: alanine dehydrogenase) by cell, just there will be NADH/NAD
+proportional imbalance.This will inevitably affect the physiological metabolism of cell and the accumulation of secondary metabolites.Azo-cycle precursor (3-amino-5-hydroxy-benzoic acid) combined coefficient of Rifamycin Sodium and the close positive correlation of vigor of glutamine synthetase (GS) also suppress by L-Ala (Ala), and cell generates Ala by alanine dehydrogenase oxidation NADH under fermentation condition, this approach flux enhancement can suppress the combined coefficient of GS activity and the amino-5-hydroxy-benzoic acid of 3-.Therefore, if the oxidation direction of NADH and efficiency under fermentation condition can be changed, that is: reduce NADH through alanine dehydrogenase catalytic route carbon flow, then can make NADH/NAD under fermentation condition
+be in imbalance state, the accumulation of Ala can be reduced again, make GS activity maintain higher level, thus promote the combined coefficient of 3-amino-5-hydroxy-benzoic acid and Rifamycin Sodium.Therefore, the suitable hydrogen acceptor of cell is selected to realize above-mentioned purpose under fermentation condition.
In early-stage Study with in producing, we determine that the concentration of ethanol in fermenting process fermented liquid is quite high, and Amycolatopsis mediterranei cell is oxidized NADH and synthesizing alcohol by ethanol dehydrogenase, and hydrogen acceptor is acetaldehyde molecule.In organic chemical industry's industry, acetaldehyde is conventional two carbon reagent, also can build heterocycle ring system; Can be used for allocating fruit essence in food service industry, also can be used for brewing wine essence, in flavoured food product, concentration is about 3.9-270mg/kg.But, do not act on the report of the New function of Amycolatopsis mediterranei as the metabolic regulation factor about external source acetaldehyde molecule, that is: the oxidative pathway of NADH is made to be moved to alcohol dehydrogenase enzyme catalysis direction by the reaction of alanine dehydrogenase catalysis, and by promoting that the activity of ethanol dehydrogenase strengthens the oxidation of NADH, the synthesis reducing L-Ala, the oxidation direction regulating NADH in fermenting process and efficiency, raising Rifamycin Sodium azo-cycle precursor (3-amino-5-hydroxy-benzoic acid) combined coefficient, promotes the report of the New function of fermentation yield of rifamycin SV.
Summary of the invention
The object of the present invention is to provide a kind of new feed process promoting Rifamycin Sodium fermentation combined coefficient, its substance is not used as the organic chemical industry two carbon reagent on conventional meaning, food service industry preparing essence by acetaldehyde, but acetaldehyde molecule is promoted oxidation migration and efficiency, the combined coefficient of enhancing Rifamycin Sodium azo-cycle precursor 3-amino-5-hydroxy-benzoic acid, the fermentation yield of raising Rifamycin Sodium of NADH in fermenting process as the metabolic regulation factor.
During the three grade fermemtation 48-72h of Amycolatopsis mediterranei, start aseptic stream and add 10h before acetaldehyde solution to fermentation ends, make its final concentration be 1.5-3.0mmol/L, at the end of cultivation, Rifamycin Sodium is tired and is on average improved 10.1%.
Described invention has the advantages that material is easily purchased, operating procedure is simple, controllability is high, be applicable to industrial fermentation production.
A kind of feed process promoting Rifamycin Sodium fermentation combined coefficient that the present invention proposes, comprises the following steps:
(1) preparation of substratum
1. seed culture medium
Glucose 1.5%, soybean cake powder 1.0%, peptone 1.0%, saltpetre 0.05%, calcium carbonate 0.2%, Zulkovsky starch 1.5%, pH7.0.For subsequent use through 0.1MPa vapor sterilization 30min;
2. fermention medium
Glucose 6-10%, fish meal 0.3-0.55%, soybean cake powder 0.5-0.65%, peptone 0.4-0.7%, saltpetre 0.6-0.8%, calcium carbonate 0.5%, potassium primary phosphate 0.02%, pH nature.For subsequent use through 0.1MPa vapor sterilization 20min.
(2) actication of culture
Amycolatopsis mediterranei (A.mediterranei) bacterial classification hidden of going bail for accesses seed culture medium, and 48h cultivated by shaking table, and culture condition is 28 DEG C, 220r/min.
(3) strain expanded culture and the collection of closing bottle seed cell
Received by above-mentioned bacterial classification in fermentation shake flask substratum, liquid amount is 50-100mL/250mL triangular flask, and inoculum size is 3%-5%, and in 27 DEG C-28 DEG C, 150-200r/min shaking culture 46-50h, shake-flask seed substratum is closed bottle by rear aseptic technique.(4) secondary seed is cultivated and three grade fermemtation
To close bottle seed flame protection access secondary seed tank, culture temperature 28 DEG C, rotating speed is 160-200r/min, and air flow control is 0.8vvm, and tank pressure controls as 0.03-0.04MPa, and after cultivating 48h, culture transferring is to three grade fermemtation tank.
(5) three grade fermemtation tank controls
Culture temperature 28 DEG C, rotating speed 0-48h is 200r/min, 48-72h be 220r/min, 72-96h be 230r/min, 96h is 240r/min to putting tank.Air flow control: 0-48h is 0.4vvm, 48-72h be 0.6vvm, 72-96h be 0.8vvm, 96h is 1vvm to putting tank.Tank pressure controls: 0-48h is 0.02MPa, 48-72h be 0.03MPa, 72-96h be 0.04MPa, 96h is 0.05MPa to putting tank.
(6), when three grade fermemtation culture cycle is 48-72h, start stream and add and fill into 10h before acetaldehyde solution to fermentation ends, make its final concentration be 1.5-3.0mmol/L, other Controlling Technology is constant.
Compared with prior art tool of the present invention has the following advantages and progress significantly: operate by the technical program, the external source acetaldehyde commonly using two carbon reagent and food service industry deodorant tune as organic chemical industry's industry is used as the metabolic regulation factor and acts on Amycolatopsis mediterranei, have adjusted oxidation migration and the efficiency of fermenting process NADH, make fermentation middle and later periods lactic acid, succsinic acid, oxysuccinic acid, the concentration of L-Ala reduces, aminoglutaric acid concentration significantly improves, more effectively facilitate the synthesis of 3-amino-5-hydroxy-benzoic acid, at the end of cultivation, Rifamycin Sodium is tired and is on average improved 10.1%.This is the most different from prior art, beyond the expection to Rifamycin Sodium facilitation effect, creates unforeseeable technique effect.
Accompanying drawing explanation
Fig. 1 be stream add acetaldehyde to biomass (zero, control group; ●: interpolation group) and Rifamycin Sodium (, control group; ■, interpolation group) effect diagram that synthesizes, data are the mean value of three parallel tests.
As shown in Figure 1: from thalline enters logarithmic phase (48h), acetaldehyde is added in constant speed flowing, its final concentration is made to be 1.5mmol/L, the fermentation later stage facilitates the growth of biomass, higher than control group 16.5%-83.7%, tire and also improved accordingly, when 121h, interpolation group potency ratio control group improves 23.3%, during fermentation ends, Rifamycin Sodium potency ratio contrast raising 10.1%.
Fig. 2 be stream add acetaldehyde to lactic acid (zero, control group; ●: interpolation group) and succsinic acid (, control group; ■, interpolation group) effect diagram that synthesizes, data are mean value ± standard deviation.
As shown in Figure 2: along with the carrying out of fermentation, stream adds lactic acid in acetaldehyde experimental group, succsinic acid is lower than the concentration of control group, particularly at 96h, the amount that stream adds lactic acid in group is 35mg/L, the concentration of succsinic acid is 36mg/L, only has 88.5% of control group respectively, and 41.9%.In microbe, glycolysis-also generates the NADH of equivalent while producing pyruvic acid, when dissolved oxygen is inadequate, the electronics that oxidation operation is released can be directly passed to substrate itself by fermentation by thalline, produce various different meta-bolites, such as lactic acid and succinate fermentative.We are after in thalline fermenting process, stream adds acetaldehyde, and its growing amount reduces, and illustrates and reduces carbon metabolism flow to organic acid compound direction after stream adds acetaldehyde, turned to alcohols compound direction on the contrary.
Fig. 3 be stream add acetaldehyde to oxysuccinic acid (zero, control group; ●: interpolation group) and L-Ala (, control group; ■, interpolation group) effect diagram that synthesizes, data are mean value ± standard deviation.
As shown in Figure 3: along with the carrying out of fermentation, the oxysuccinic acid that stream adds in acetaldehyde experimental group is lower than the concentration of control group, particularly at 96h, the concentration that stream adds group oxysuccinic acid is 138mg/L, only has control group 55.4%.In microbe, glycolysis-also generates the NADH of equivalent while producing pyruvic acid, when dissolved oxygen is inadequate, the electronics that oxidation operation is released can be directly passed to substrate itself by fermentation by thalline, produce various different meta-bolites, such as: oxysuccinic acid acid-fermentation.We are after in thalline fermenting process, stream adds acetaldehyde, and its growing amount reduces, and illustrates and reduces carbon metabolism flow to organic acid compound direction after stream adds acetaldehyde, turned to alcohols compound direction on the contrary.In addition, along with the carrying out of fermentation, it is lower than the concentration of control group that stream adds L-Ala in group, 112h, and it is 5.6mg/L that stream adds group L-Ala concentration, only has 26.3% of control group.Illustrate that stream adds the exogenous electron acceptor of acetaldehyde as NADH, be reduced into ethanol, thus reduce the transformation efficiency of pyruvic acid to L-Ala, and the activity of L-Ala and glutamine synthetase (GS) inversely, when L-Ala concentration reduces, GS enzyme is lived and is risen, and the amount of L-glutamic acid also adds accordingly, more L-glutamic acid is made to change into glutamine under the effect of GS enzyme, the amide nitrogen of glutamine is again 3-amino-5-hydroxy-benzoic acid (AHBA) directly donor, thereby promoting the synthesis of Rifamycin Sodium.
Fig. 4 be stream add acetaldehyde to L-glutamic acid (zero, control group; ●: interpolation group) and phenylalanine (, control group; ■, interpolation group) effect diagram that synthesizes, (data are mean value ± standard deviation).
As shown in Figure 4: along with the carrying out of fermentation, it is higher than the concentration of control group that stream adds group Glutamic Acid, 112h, and it is 2.3g/L that stream adds group aminoglutaric acid concentration, than contrast raising 29%; And the phenylalanine that stream adds group after fermentation the phase lower than contrast, during 120h, the concentration that stream adds group phenylalanine is 0.238mg/L, only has 17.1% of contrast.The exogenous electron acceptor of acetaldehyde as NADH is added from our analysis stream of above result, ethanol is reduced into by it, thus reduce the transformation efficiency of pyruvic acid to L-Ala, and the activity of L-Ala and glutamine synthetase (GS) inversely, when L-Ala concentration reduces, GS enzyme is lived and is risen, and the amount of L-glutamic acid also adds accordingly, more L-glutamic acid is made to change into glutamine under the effect of GS enzyme, the amide nitrogen of glutamine is again 3-amino-5-hydroxy-benzoic acid (AHBA) directly donor, thereby promoting the synthesis of Rifamycin Sodium.
Embodiment
Embodiment 1
1, the preparation of substratum
(1) seed culture medium
Glucose 1.5g, soybean cake powder 1.0g, peptone 1.0g, saltpetre 0.05g, calcium carbonate 0.2g, Zulkovsky starch 1.5g, is settled to 100mL, pH7.0.For subsequent use through 0.1MPa autoclaving 30min;
(2) fermention medium
Glucose 6-10g, fish meal 0.3-0.55g, soybean cake powder 0.5-0.65g, peptone 0.4-0.7g saltpetre 0.6-0.8g, calcium carbonate 0.5g, potassium primary phosphate 0.02g, is settled to 100mL, pH nature.For subsequent use through 0.1MPa autoclaving 30min;
2, actication of culture
Amycolatopsis mediterranei (A.mediterranei) bacterial classification hidden of going bail for accesses seed culture medium, and 48h cultivated by shaking table, and culture condition is 28 DEG C, 220r/min;
3, the collection of strain expanded culture and conjunction bottle cell
Received by above-mentioned bacterial classification in fermentation shake flask substratum, liquid amount is 50mL/250mL triangular flask, and inoculum size is 5%, and in 27 DEG C, 150r/min shaking culture 46h, then shake-flask seed substratum is closed bottle by aseptic technique;
4, secondary seed is cultivated and three grade fermemtation
To close bottle seed flame protection access secondary seed tank, culture temperature 28 DEG C, rotating speed is 160r/min, and air flow control is 0.8vvm, and tank pressure controls to fill into three grade fermemtation tank for 0.03MPa, 48h aseptic technique;
5, three grade fermemtation tank controls
Culture temperature 28 DEG C, rotating speed 0-48h is 200r/min, 48-72h be 220r/min, 72-96h be 230r/min, 96h is 240r/min to putting tank.Air flow control: 0-48h is 0.4vvm, 48-72h be 0.6vvm, 72-96h be 0.8vvm, 96h is 1vvm to putting tank.Tank pressure controls: 0-48h is 0.02MPa, 48-72h be 0.03MPa, 72-96h be 0.04MPa, 96h is 0.05MPa to putting tank.
6, the adding of regulatory factor
Commercially available acetaldehyde aseptic technique mixes with sterilized water, and during fermentation period 48h, start aseptic technique and acetaldehyde solution stream is added to three grade fermemtation tank (100L), to fermentation ends, 10h stops stream adding, and makes acetaldehyde final concentration be 1.5mmol/L.
Embodiment 2
1, the preparation of substratum: with embodiment 1.
2, actication of culture: with embodiment 1.
3, the collection of strain expanded culture and conjunction bottle cell: with embodiment 1.
4, secondary seed is cultivated and three grade fermemtation: with embodiment 1.
5, three grade fermemtation tank controls: with embodiment 1.
6, regulatory factor is added
Commercially available acetaldehyde aseptic technique mixes with sterilized water, and when fermentation period is 60h, starts aseptic technique and acetaldehyde solution stream is added to three grade fermemtation tank (100L), to fermentation ends, 10h stops stream adding, and makes acetaldehyde final concentration be 2.0mmol/L.
Embodiment 3
1, the preparation of substratum: with embodiment 1.
2, actication of culture: with embodiment 1.
3, the collection of strain expanded culture and conjunction bottle cell: with embodiment 1.
4, secondary seed is cultivated and three grade fermemtation: with embodiment 1.
5, three grade fermemtation tank controls: with embodiment 1.
6, regulatory factor is added
Commercially available acetaldehyde aseptic technique mixes with sterilized water, and when fermentation period is 72h, starts aseptic technique and acetaldehyde solution stream is added to three grade fermemtation tank (100L), to fermentation ends, 10h stops stream adding, and makes acetaldehyde final concentration be 3.0mmol/L.
Claims (1)
1. one kind promotes the feed process of Rifamycin Sodium fermentation combined coefficient, it is characterized in that: Amycolatopsis mediterranei carries out three grade fermemtation cultivation, when the three grade fermemtation cycle at 48-72h time, start aseptic stream in culture and add 10h before acetaldehyde solution to fermentation ends, make acetaldehyde final concentration be 1.5-3.0mmol/L.
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Cited By (2)
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| CN105861410A (en) * | 2016-05-26 | 2016-08-17 | 湖北工业大学 | Method for improving clostridium butyricum growth efficiency |
| CN112410270A (en) * | 2020-12-14 | 2021-02-26 | 宁夏泰胜生物科技有限公司 | Culture medium and culture method for producing rifamycin by fermenting amycolatopsis mediterranei |
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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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| 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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Cited By (3)
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| CN105861410A (en) * | 2016-05-26 | 2016-08-17 | 湖北工业大学 | Method for improving clostridium butyricum growth efficiency |
| CN105861410B (en) * | 2016-05-26 | 2019-05-24 | 湖北工业大学 | A method of improving Miyarisan growth efficiency |
| CN112410270A (en) * | 2020-12-14 | 2021-02-26 | 宁夏泰胜生物科技有限公司 | Culture medium and culture method for producing rifamycin by fermenting amycolatopsis mediterranei |
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