CN103642870A - Fermentation production method of rifamycin SV based on oxygen uptake rate OUR used as control parameter - Google Patents
Fermentation production method of rifamycin SV based on oxygen uptake rate OUR used as control parameter Download PDFInfo
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Abstract
The invention discloses a fermentation production method of rifamycin SV based on oxygen uptake rate OUR used as a control parameter. By using nocardia intermedien as an original strain, rifamycin SV is produced by virtue of pure culture and three-level fermentation. In the fermentation preparation process, the stirring rotation speed of a fermentation tank and ventilation flow in the fermentation process are controlled to reach different oxygen supply levels; the oxygen uptake rate OUR of a fermentation system at high level is kept to be between 18 and 20mmol/L/h in 70-75 hours in the earlier stage of the fermentation process, so that hypha is quickly grown and the titer is quickly increased; after 70-75 hours in the earlier stage of the fermentation process, the stirring rotation speed of the fermentation tank and the ventilation flow are reduced, and the oxygen uptake rate OUR is controlled to be between 13 and 15mmol/L/h until the tank is put at the end. According to the method, the thallus growth and the synthesis of the rifamycin SV are increased, the conversion rate is increased, the production period is shortened, the fermentation production level is stably improved, and the yield of the rifamycin SV is improved.
Description
Technical field
The present invention relates to a kind of fermentation method for producing of Rifamycin Sodium, specifically a kind of is the fermentation method for producing of controlling the Rifamycin Sodium of parameter based on oxygen consumption rate OUR, special relevant with the process control in Rifamycin Sodium fermentative production.
Background technology
Rifamycinoid antibiotics is the class microbiotic being produced by Mediterranean Sea Streptothrix, it has broad-spectrum antibacterial action, all very strong to gram positive bacteriums, the particularly effect of resistance streptococcus aureus such as tubercule bacillus, leprosy bacillus, suis, streptococcus pneumoniae.Also effective to some Gram-negative bacteria.Rifamycin drug has: rifamide, Rifampin etc.At present in clinical application, there are Rifampin, rifapentine and a rifabutin.The direct precursor of these medicines is all Rifamycin Sodium.At existing Rifamycin Sodium, criticize in cultivation and fermentation technique, mostly only adopt pH, DO, temperature etc. as foundation and the means of control strategy.These parameter most applications only can reflect performance or the operational condition of equipment, and physiological status can not be real-time, that reflect really microorganism.Rifomycin fermenting process belongs to oxygen consumption fermentation, and oxygen supply is being formed with important impact to producing growth and the product of bacterial strain.In rifomycin fermenting process, suitable stirring and ventilation must be provided, required meta-bolites could be bred and accumulate to thalline.In addition, in the oxygen requirement of the thalline in different fermentations stage, be different, the size of the oxygen delivery capacity of fermented liquid directly affects activity, pathways metabolism and the product output of the enzyme of microorganism.Therefore study oxygen supply size to the impact of fermentation and control enhancing productivity, improving quality product etc. all significant.General oxygen consumption fermenting process is all controlled higher oxygen supply to avoid the generation of oxygen restriction, in this case can be with dissolved oxygen concentration (Dissolved Oxygen, be called for short DO) characterize Oxygen supplied level, by controlling mixing speed and air flow quantity, can effectively control DO.Dissolved oxygen level is subject to oxygen supply speed (OTR) and oxygen consumption rate (OUR) two aspect impacts.Wherein the influence factor of OTR mainly contains rheological characteristics and the operating parameters of fermented liquid, such as flow, mixing speed, stirring arm type etc.Different from DO and OTR, OUR is a typical physiological parameter, can reflect the real oxygen consumption situation of microorganism.As a rule, more than fermentation generally can be controlled at critical dissolved oxygen by dissolved oxygen level, otherwise oxygen supply will become restrictive factor, and OUR will change along with the variation of OTR, and this situation is generally unfavorable for fermenting and carries out.Another important physiological parameter is respiratory quotient (RQ), and RQ is the ratio of release of carbonate dioxide speed (CER) and OUR.RQ can change along with the variation of pathways metabolism and flux, be exactly in other words, by RQ can the monitor fermentation process of real-time online in the metabolism state of microorganism.
The Rifamycin Sodium of the having reported relevant technical study of fermenting mainly concentrates on the production level that improves rifomycin by genetic engineering modified technology, or by induction mutation of bacterium process, single factors optimization regulation experiment of simple nitrogenous source and phosphate concn investigates its impact synthetic on product, the patent literature also having adopts optimizes fermentation production process by the feed rate of adjustment process.These technology are just studied the impact on Rifamycin Sodium fermentation from monofactorial investigation angle, certainly for certain effect has also been played in the lifting of production level, but these researchs lack from the angle analysis bio-metabolic process of the dynamic physiology metabolic process of thalline, all do not find suitable way investigate with controlled fermentation process on the most important oxygen consumption level of Rifamycin Sodium fermentation impact, to reach the object that improves production capacity.Oxygen level of supply not enough or excessive in fermentation production process is controlled, and can have a strong impact on the stability of production technique, brings the problems such as production cost is high, productive rate is low, qualification rate is low.
By retrieval, contriver has found the fermentation process that following Patents document: CN103184260A discloses a kind of high-content berythromycin fermented liquid, this fermentation process is to take streptomyces erythreus in the process of strain fermentation production erythromycin, and the air flow of controlled fermentation liquid and mixing speed make the dissolved oxygen content of fermented liquid remain on 0 ~ 15%.By 31 ~ 37 ℃ of temperature, pH is in 6.7 ~ 7.3 fermentation system, ventilation ratio and mixing speed that control passes into sterile air carry out the dissolved oxygen content in controlled fermentation process, pathways metabolism is changed, thereby effectively improve the accumulation of berythromycin, the content that improves berythromycin in fermented liquid, for purification of erythromycin, B supplies raw materials.CN102660598A discloses a kind of method that improves Rifamycin Sodium fermentation yield.In the process of fermentation culture, the oxalate that is 0.2 ~ 0.5g/L by final concentration, the soya-bean oil of 2 ~ 5mL/L is aseptic fills into the fermentation culture Amycolatopsis mediterranei cycle in the fermentor tank of 60 ~ 80h.CN102703541A discloses a kind of feed supplement method that improves Rifamycin Sodium fermentation yield, in the process of fermentation culture, the L-Ala that the fosfomycin sodium of the L-Ala that is 0.3 ~ 0.8g/L by final concentration, the soya-bean oil of 2 ~ 5mL/L and 3 ~ 20mg/L or final concentration are 0.3 ~ 0.8g/L, the soya-bean oil of 2 ~ 5mL/L and the fosfomycin trometamol of 3 ~ 20mg/L are aseptic fills into the fermentation culture Amycolatopsis mediterranei cycle in the three grade fermemtation tank of 60 ~ 80h, and while cultivating end, Rifamycin Sodium is tired and on average improved 17.0%.How these patent documentations are stablized raising fermentative production level, the output of Rifamycin Sodium are improved for the present invention above, do not provide concrete technical director's scheme.
Summary of the invention
Technical problem to be solved by this invention is, provide a kind of based on oxygen consumption rate OUR for controlling the fermentation method for producing of the Rifamycin Sodium of parameter, this production method can be stablized raising fermentative production level, and the output of Rifamycin Sodium is improved.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of is the fermentation method for producing of controlling the Rifamycin Sodium of parameter based on oxygen consumption rate (oxygen uptake rate) OUR, as starting strain by Nocardia intermedien, through purebred cultivation, three grade fermemtation generates Rifamycin Sodium, concrete technology step comprises the preparation section on inclined-plane, female bottle seed preparation section, first order seed preparation section, secondary seed preparation section, fermentation preparation section, its technical scheme is: at fermentation preparation section, mixing speed and ventilation flow rate (ventilation ratio) by fermentor tank in controlled fermentation process reach different Oxygen supplied levels, by regulating mixing speed, ventilation recently realizes the accurate control of oxygen consumption level, specifically at fermenting process, in 70 ~ 75 hours early stages, maintain the high-caliber oxygen consumption rate OUR of fermentation system between 18 ~ 20mmol/L/h, make mycelia Fast Growth, tire and raise fast, after fermenting process 70 ~ 75 hours used times of early stage, by reducing mixing speed and the ventilation flow rate of fermentor tank, oxygen consumption rate OUR is controlled between 13 ~ 15mmol/L/h, until put tank terminal (fermentation termination).Thereby promote the synthetic of thalli growth and Rifamycin Sodium, promote transformation efficiency and shorten the production cycle.
In technique scheme, oxygen consumption velocity level's reduction in described fermenting process (reduction of oxygen consumption rate) preferably stage reduction is controlled, is declined continuously or progressively reduce.Above-mentioned fermenting process maintains the high-caliber oxygen consumption rate OUR of fermentation system in 70 ~ 75 hours early stages and is preferably 19.1mmol/L/h.After above-mentioned fermenting process 70 ~ 75 hours used times of early stage, oxygen consumption rate OUR is preferably controlled to 14.2mmol/L/h.Product resultant quantity >=7300mg/L ~ the 7500mg/L of above-mentioned Rifamycin Sodium.
The inventor controls different Oxygen supplied levels by mixing speed different in controlled fermentation process and ventilation flow rate.Experiment is carried out at 50L bench scale.
Instrument and reagent:
Fermentor tank: the biochemical equipment 15L of limited liability company of Shanghai Guoqiang and 50L Fermentation;
Instrument: ExtralMAX30-LG process mass spectrograph; Country's biochemical engineering center Biostar software package; 722 type ultraviolet one visible spectrophotometers; Rotary shaking table.
Cultural method: rifomycin fermentation adopts three grade fermemtation, ferments comprising secondary seed.
Female bottle seed culture: fill seed culture medium 100mL in 1L triangular flask, after inclined plane inoculating, shaking table is cultivated, 28 ℃ of culture temperature, rotating speed 220rpm, cultivates approximately 48 hours.
Seeding tank: loading amount 8L in 15L seeding tank, mother's bottle seed is pressed to the inoculation of 10%-15% inoculum size, 28 ~ 29 ℃ of culture temperature, control dissolved oxygen level with rotating speed and air air and are not less than 35%, and natural pH controls, culture cycle approximately 50 hours.
Fermentor tank: 50L fermentor tank loading amount 30L, with the pipeline culture transferring 5L from 15L seeding tank through vapor sterilization, temperature is controlled at 26 ~ 29 ℃, with rotating speed and ventilation, controlling OUR level is 18mmol/L/h, after 70 hours, OUR level is 15mmol/L/h, nature pH controls, and adds glucose and water, culture cycle approximately 130 ~ 140 hours in process.
Measuring method:
Reducing sugar test: DNS method.
Biomass estimation: determined off-line adopts humid volume method, and 10mL fermented liquid is placed in to centrifuge tube, and the centrifugal 15min of 3000rpm, pours centrifugal supernatant into graduated cylinder, calculates the volume of fermented liquid according to the volumeter of supernatant.The online viable cell sensor that adopts U.S. Aber company, its principle is polarized under electric field according to cell, forms a plurality of electric capacity, the size of capacitance is directly proportional to viable bacteria amount.So, can adopt capacitance size to estimate the amount of viable bacteria.
Titration: adopt state-promulgated pharmacopoeia method.
PH, DO on-line determination: adopt the high temperature resistant electrode of Mettler Toledo to carry out on-line determination.
Temperature: platinum temperature electrode on-line determination.
The mensuration of oxygen and carbon dioxide in air inlet and tail gas: adopt U.S. Extrel process mass spectrum MAX300-LG to carry out real-time online collection analysis to the air inlet in fermenting process and tail gas.
Oxygen consumption rate OUR and carbonic acid gas generating rate CER measure: the calculating of OUR and CER calculates by the analytical data to fermentation tail gas.With rare gas element N in air inlet and tail gas
2maintain constant equilibrium establishment equation, the calculation formula of trying to achieve OUR and CER is as follows:
F wherein
infor charge flow rate, the mmol/L/h of unit; V is fermentating liquid volume, the L of unit; O
2in, O
2outfor oxygen volumetric molar concentration in air inlet and tail gas, CO
2in, C
-o
2outfor carbonic acid gas volumetric molar concentration in air inlet and tail gas; N
2in,
-n
2outfor nitrogen volumetric molar concentration in air inlet and tail gas.
In 50L fermentor tank, carry out rifomycin fermentation, by regulating the oxygen uptake rate OUR level of thalline to optimize the impact of Oxygen supplied level on product synthesis rate.This research is first in the fermenting process of Rifamycin Sodium, by adjusting ventilation flow rate and mixing speed, control different Oxygen supplied levels, studied and produced the metabolic condition of bacterium under different oxygen consumption rate states, in whole process, by adding glucose, maintain certain remaining sugar concentration, make whole fermenting process in non-sugared restriction state.As can be seen from Figure 1, the oxygen consumption rate OUR of the higher thalline of Oxygen supplied level is also higher.But, it should be noted that: after 500rpm condition bottom fermentation proceeds to 100 hours, by mending sugar, can not maintain OUR, present downtrending.This is because too high OUR has impelled the aging of mycelia, by microscopic examination, also confirmed that this (see figure 3): 500rpm fermented to about 100 hours, mycelia is difficult for being colored and mycelia is rarer falls apart, and the same cycle of 300rpm fermentation, mycelia dyeing is darker, also more encrypts.
It is higher that Fig. 2 demonstrates OUR, the growth of tiring at initial stage is faster, but high OUR tired and can no longer increase even appearance decline to the later stage, while especially fermenting by about 100 hours when 500rpm, not only OUR cannot continue to maintain, and the phenomenon that also appearance significantly declines of tiring.By online viable cell sensor, recording bacterium amount is also very identical with the variation tendency of OUR.
Table 1 is the sugared ratio of consumption of tiring under differing temps, and related data is in Table 1.
From table 1, show and can find out, the sugar yield rule of tiring under differing temps, OUR is lower, and sugar yield is higher, but rotating speed reaches after 500rpm, and due to the decline that the later stage tires, sugar yield sharply declines.
The fact based on above, rifomycin novel process adopts two sections of OUR control strategies, uses early stage higher OUR make mycelia Fast Growth and produce and tire fast, and the later stage is reduced OUR level, and minimizing consumption sugar and mycelia are aging, obtain the balance of consumption sugar and productive rate.In Fig. 4, shown that OUR controls stage by stage, control OUR at 18-20mmol/L/h early stage, the Fast Growth of tiring, to 70h left and right, by reducing rotating speed, OUR is controlled to 15mmol/L/h, tire and continue to increase, finally tire and reach about 6600mg/L, but the cycle but shorten to 113h, the consumption of tiring sugar is than 57mg RSV/g glucose.
Simple two-part temperature is controlled OUR strategy still can cause finally cannot keeping the level of final stage OUR, after OUR declines, can find out that the rate of growth of tiring slows down.In the total fermentation time that approaches 1/3, only produced approximately 15% total titer.Therefore apply OUR-rotating speed interlock control strategy and carry out Rifamycin Sodium fermenting process on-line Control.As shown in Figure 5.Fermentation is carried out with 500rpm the initial stage, in fermenting process, by reducing rotating speed, maintains early stage OUR not higher than 16mmol/L/h, later stage OUR slow decreasing.Result has maintained and has approached 120 hours at fermenting process, tires and reaches 7400mg/L.The consumption sugar amount that unit tires is consistent with two-stage control strategy before.Next test is controlled OUR at different levels by the interlock of OUR-rotating speed, cannot maintain present level start to drop to fermentation ends with OUR.Find that OUR maintains below 300rpm in the following rotating speed whole process of 10mmol/L/h, the cycle is generally longer.In 10-15mmol/L/h change procedure, along with the change of OUR level is large, the cycle shortens fast, finally tires and changes not quite, tires slightly on a declining curve to sugared productive rate.And when OUR maintains high level, early stage, cultivation results was more consistent, finally tire and start to decline, sugared productive rate is sharply declined, and final remaining sugar concentration raises to some extent.Finally determined that usining OUR-rotating speed interlock controls the rifomycin fermenting process as optimum controling strategy, output is brought up to 7400mg/L by 6500U/10mL, and the cycle shortened to 120 hours by 135 hours simultaneously.
In sum, the present invention has promoted the synthetic of thalli growth and Rifamycin Sodium, has promoted transformation efficiency, has shortened the production cycle.Stablize and improved fermentative production level, the output of Rifamycin Sodium is improved.
Accompanying drawing explanation
Fig. 1 is under different Oxygen supplied levels (under different fermentor tank mixing speed and ventilation flow rate), the graphic representation of the oxygen consumption rate OUR of each fermenting process.
Fig. 2 is under different Oxygen supplied levels (under different fermentor tank mixing speed and ventilation flow rate), the valence value graphic representation of each fermenting process.
Fig. 3 is under different Oxygen supplied levels (being different OUR), and fermenting process was at the mycelia aspect graph of 100 hours.
Fig. 4 is the graphic representation that oxygen consumption rate OUR controls stage by stage.
Fig. 5 is that oxygen consumption rate OUR is at the graphic representation of mixing speed and ventilation flow rate interlock control.
Embodiment
As shown in Figure 1, the curve of triangle sign under black in Fig. 1, its corresponding mixing speed is 500rpm, air flow is 1vvm; The curve of triangle sign on black, its corresponding mixing speed is 400rpm, air flow is 1vvm; The curve of the circular sign of black, its corresponding mixing speed is 300rpm, air flow is 0.8vvm; The curve of black square sign, its corresponding mixing speed is 200rpm, air flow is 0.6vvm.
As shown in Figure 2, the curve of triangle sign under black in Fig. 2, its corresponding mixing speed is 500rpm, air flow is 1vvm; The curve of triangle sign on black, its corresponding mixing speed is 400rpm, air flow is 1vvm; The curve of the circular sign of black, its corresponding mixing speed is 300rpm, air flow is 0.8vvm; The curve of black square sign, its corresponding mixing speed is 200rpm, air flow is 0.6vvm.
As shown in Figure 3, Fig. 3 is microscopic examination photo: left-hand component mixing speed is 300rpm, and right-hand component mixing speed is 500rpm.
As shown in Figure 4, this figure is the graphic representation that oxygen consumption rate OUR controls stage by stage, the curve display oxygen consumption rate OUR of triangle sign on black in figure, the curve display flow of the circular sign of black, the curve display rotating speed of black square sign, under black, the curve display RSV of triangle sign tires.
As shown in Figure 5, this figure is that oxygen consumption rate OUR is at the graphic representation (same Fig. 4) of mixing speed and ventilation flow rate interlock control.
Embodiment 1: the fermentation method for producing of Rifamycin Sodium of the present invention is as starting strain by Nocardia intermedien, the purebred cultivation of process, three grade fermemtation generate Rifamycin Sodium, and concrete technology step comprises the preparation section on inclined-plane, female bottle seed preparation section, first order seed preparation section, secondary seed preparation section, fermentation preparation section.Except fermentation preparation section, other each operation is prior art.The further processing step of each operation of the present invention is as follows:
(1), the preparation section on inclined-plane: 1. female inclined-plane preparation: by aseptic technique requirement, with the appropriate sand seed of inoculating needle picking, be evenly coated on slant medium, require even, sparse, be convenient to select, in 27.5 ± 0.5 ℃ of constant temperature culture 9 ~ 10 days, after growth and maturity or growth normal, be kept in 2 ~ 5 ℃ of refrigerators standby.2. sub-inclined-plane preparation: by aseptic requirement, select female inclined-plane bacterium colony of having grown with inoculating needle, evenly line on blank substratum, mycelia will be evenly distributed, in 27.5 ± 0.5 ℃ of constant temperature culture 6 ~ 7 days, is stored in 2 ~ 5 ℃ of refrigerators interior standby after maturation.(2), female bottle seed preparation section: select qualified production inclined-plane seed, with inoculation shovel shovel, getting size is the well-grown inclined-plane of 1 * 23cm mycelia, be inoculated in female bottle substratum, be placed on rotating speed 220 ~ 240r/min bottle swingging machine, under 27.5 ± 0.5 ℃ of conditions, cultivate 48 hours, select 7 bottles of the normal same bacterial classification of visual appearance number and merge into 1 bottle of female bottle seed.(3), first order seed preparation section: mother's bottle seed is inoculated in first class seed pot with flame method, and inoculation is rear by 1: 0.65vvm(V/Vmin) ventilation ratio passes into sterile air, 30 ~ 28 ℃ of tank temperature, tank pressure 0.05Mpa, 48 ~ 52 hours cycles.(4), secondary seed preparation section: secondary seed tank adopts real tank sterilization, sterilization pressure 0.1Mpa, 120 ℃ of temperature, sterilizing 30 minutes.After sterilization, when tank temperature drop to 30 ℃, cultured first order seed is moved in secondary seed tank, after culture transferring, pass into sterile air, by 1: the air flow of 0.78vvm ventilation ratio, tank pressure 0.04 ~ 0.05Mpa, 28 ~ 30 ℃ of temperature, cultivate 48 ~ 52 hours.(5), fermentation preparation section: at fermentation preparation section, contain in every liter of substratum aqueous solution in fermentor tank (50L fermentor tank loading amount 30L) (comprising in other words): 70 grams of glucose, 5 grams of fish meal, 8 grams, saltpetre, 8 grams of peptones, 4 grams of medicine matchmakers, 12 grams of soybean cake powders, 5 grams, calcium carbonate, 0.2 ~ 0.3 gram of potassium primary phosphate, 1 ~ 1.29 milliliter of bubble enemy, 1 milligram of cobalt chloride.Fermentor tank sterilization employing Carbon and nitrogen sources divides disappear (sterilization separately of glucose, potassium primary phosphate can being put together, this is prior art), then closes tank.The feeding mode of above-mentioned each component is that above-mentioned each component is put into fermentor tank.When fermentation jar temperature is down to 30 ℃, cultured secondary seed is moved into fermentor tank, culture transferring secondary fermentation tank fermentative production is taked alternating temperature control, and (temperature is carried out wave band control, temperature is controlled at 30 ~ 25 ℃), 0 ~ 40 hour temperature is 28 ~ 30 ℃ (can select 29 ℃), after 40 hours, temperature is 25 ~ 27 ℃ (can select 26 ℃), until put tank, the operating pressure of fermentor tank is that 0.01 ~ 0.03Mpa(can select 0.02Mpa), fermentor tank ventilation ratio (being by the sterile air volume ratio aseptic compressed air consumption of unit volume nutrient solution in per minute) is: 1: 0.6 ~ 1vvm ventilation ratio, fermentation period is 120 ~ 133 hours.Within every 8 hours, get aseptic sample and detect that pH value, sugar, bacterium are dense, microscopy mycelia, after 40 hours, start survey and tire, within 90 hours, start to survey filtering velocity, after 100 hours, stop getting aseptic sample.After having fermented, delivering to refinement workshop extracts.
At this fermentation preparation section, the mixing speed of fermentor tank adopts OUR-rotating speed interlock control strategy to carry out Rifamycin Sodium fermenting process on-line Control.Namely mixing speed and the ventilation flow rate (ventilation ratio) by fermentor tank in controlled fermentation process reaches different Oxygen supplied levels, by regulating mixing speed, ventilation recently realizes the accurate control of oxygen consumption level, specifically at fermenting process, in 70 ~ 72 hours early stages, (mixing speed carries out with 500rpm, fermentor tank ventilation ratio is 1: 0.8 ~ 0.9vvm) maintain the high-caliber oxygen consumption rate OUR of fermentation system (for 19.1mmol/L/hr) between 18 ~ 20mmol/L/hr, make mycelia Fast Growth, tire and raise fast, after fermenting process 70 ~ 72 hours used times of early stage, by reducing the mixing speed of fermentor tank and ventilation flow rate, (mixing speed carries out with 280 rpm, fermentor tank ventilation ratio is 1: 0.6 ~ 0.7vvm), oxygen consumption rate OUR is controlled to (for 14.2mmol/L/hr) between 13 ~ 15mmol/L/hr, until put tank terminal (fermentation termination).Thereby promote the synthetic of thalli growth and Rifamycin Sodium, promote transformation efficiency and shorten the production cycle.Ultimate capacity is brought up to 7400mg/L by 6500mg/L, and the cycle shortened to 120 hours by 135 hours simultaneously.In above-mentioned fermenting process, oxygen consumption velocity level's reduction is that the reduction of oxygen consumption rate is that stage reduction is controlled, declined continuously or progressively reduce.Product resultant quantity >=7300mg/L ~ the 7500mg/L of above-mentioned Rifamycin Sodium.
The present invention is the Rifamycin Sodium process control method that a kind of OUR of take be to instruct, by Rifamycin Sodium fermenting process to the on-line analysis of tail gas and software package in line computation, determined a kind of for instructing rifomycin control strategy.First experiment carries out on-line parameter collection to existing fermenting process, not only comprise the operational variables such as conventional pH, dissolved oxygen, temperature, ventilation, stirring, also comprise the physiological parameters such as OUR, CER, RQ, and adopt viable cell sensor to carry out on-line monitoring to viable bacteria amount.By the OUR under oxygen restricted condition, regulate and control, finally make the output of rifomycin be improved, also developed a kind of optimization method simultaneously.
Embodiment 2: the fermentation method for producing of Rifamycin Sodium of the present invention is as starting strain by Nocardia intermedien, the purebred cultivation of process, three grade fermemtation generate Rifamycin Sodium, and concrete technology step comprises the preparation section on inclined-plane, female bottle seed preparation section, first order seed preparation section, secondary seed preparation section, fermentation preparation section.At fermentation preparation section, mixing speed and ventilation flow rate by fermentor tank in controlled fermentation process reach different Oxygen supplied levels, specifically at fermenting process, in 74 ~ 75 hours early stages, (mixing speed carries out with 550rpm, fermentor tank ventilation ratio is 1: 0.8 ~ 0.9vvm) to maintain the high-caliber oxygen consumption rate OUR of fermentation system be 20mmol/L/h(or 18 mmol/L/h), make to tire and raise fast, after fermenting process 74 ~ 75 hours used times of early stage, by reducing the mixing speed of fermentor tank and ventilation flow rate, (mixing speed carries out with 300rpm, fermentor tank ventilation ratio is 1: 0.6 ~ 0.7 vvm), oxygen consumption rate OUR is controlled to 15mmol/L/h(or 13 mmol/L/h), until put tank terminal.In above-mentioned fermenting process, oxygen consumption velocity level's reduction is that the reduction of oxygen consumption rate is that stage reduction is controlled, declined continuously or progressively reduce.Product resultant quantity >=7300mg/L ~ the 7500mg/L of above-mentioned Rifamycin Sodium.
Claims (5)
1. one kind is the fermentation method for producing of controlling the Rifamycin Sodium of parameter based on oxygen consumption rate OUR, as starting strain by Nocardia intermedien, the purebred cultivation of process, three grade fermemtation generate Rifamycin Sodium, concrete technology step comprises the preparation section on inclined-plane, female bottle seed preparation section, first order seed preparation section, secondary seed preparation section, fermentation preparation section, it is characterized in that:
At fermentation preparation section, mixing speed and ventilation flow rate by fermentor tank in controlled fermentation process reach different Oxygen supplied levels, specifically at fermenting process, in 70 ~ 75 hours early stages, maintain the high-caliber oxygen consumption rate OUR of fermentation system between 18 ~ 20mmol/L/h, make to tire and raise fast, after fermenting process 70 ~ 75 hours used times of early stage, by reducing mixing speed and the ventilation flow rate of fermentor tank, oxygen consumption rate OUR is controlled between 13 ~ 15mmol/L/h, until put tank terminal.
2. the fermentation method for producing of Rifamycin Sodium according to claim 1, the reduction that it is characterized in that oxygen consumption velocity level in above-mentioned fermenting process is that the reduction of oxygen consumption rate is that stage reduction is controlled, declined continuously or progressively reduce.
3. the fermentation method for producing of Rifamycin Sodium according to claim 1, is characterized in that it is 19.1mmol/L/h that above-mentioned fermenting process maintains the high-caliber oxygen consumption rate OUR of fermentation system in 70 ~ 75 hours early stages.
4. the fermentation method for producing of Rifamycin Sodium according to claim 1, after it is characterized in that above-mentioned fermenting process 70 ~ 75 hours used times of early stage, is controlled at 14.2mmol/L/h by oxygen consumption rate OUR.
5. the fermentation method for producing of Rifamycin Sodium according to claim 1, is characterized in that the product resultant quantity >=7300mg/L ~ 7500mg/L of above-mentioned Rifamycin Sodium.
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| CN105154490A (en) * | 2015-09-22 | 2015-12-16 | 江苏威凌生化科技有限公司 | Dectomax fermentation production method with OUR as control parameter |
| CN110551783A (en) * | 2019-09-23 | 2019-12-10 | 华东理工大学青岛创新研究院 | Fermentation method for producing gentamicin by using continuous flow feeding material with oxygen consumption rate as key control parameter |
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|---|---|---|---|---|
| CN104357586A (en) * | 2014-11-11 | 2015-02-18 | 河北欣港药业有限公司 | Fermentation production method of rifamycin SV based on phosphate glycine betaine concentration as control parameter |
| CN104357586B (en) * | 2014-11-11 | 2015-11-04 | 河北欣港药业有限公司 | It is the fermentation method for producing of the Rifamycin Sodium of controling parameters based on phosphoric acid betaine concentration |
| CN105154490A (en) * | 2015-09-22 | 2015-12-16 | 江苏威凌生化科技有限公司 | Dectomax fermentation production method with OUR as control parameter |
| WO2017049751A1 (en) * | 2015-09-22 | 2017-03-30 | 江苏威凌生化科技有限公司 | Dectomax fermentation production method on basis of using oxygen uptake rate (our) as control parameter |
| CN110551783A (en) * | 2019-09-23 | 2019-12-10 | 华东理工大学青岛创新研究院 | Fermentation method for producing gentamicin by using continuous flow feeding material with oxygen consumption rate as key control parameter |
| CN110551783B (en) * | 2019-09-23 | 2021-07-06 | 华东理工大学青岛创新研究院 | Fermentation method for producing gentamicin by using continuous flow feeding material with oxygen consumption rate as key control parameter |
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| CN103642870B (en) | 2014-11-19 |
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