WO2017049751A1 - 基于耗氧速率our为控制参数的多拉菌素发酵生产方法 - Google Patents

基于耗氧速率our为控制参数的多拉菌素发酵生产方法 Download PDF

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WO2017049751A1
WO2017049751A1 PCT/CN2015/094999 CN2015094999W WO2017049751A1 WO 2017049751 A1 WO2017049751 A1 WO 2017049751A1 CN 2015094999 W CN2015094999 W CN 2015094999W WO 2017049751 A1 WO2017049751 A1 WO 2017049751A1
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fermentation
oxygen
doramectin
rate
oxygen consumption
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PCT/CN2015/094999
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凌青云
李林
张义文
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江苏威凌生化科技有限公司
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    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin

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  • the invention relates to the field of doramectin production, in particular to a method for fermentative production of doramectin based on an oxygen consumption rate OUR as a control parameter.
  • Doramectin the trade name "Dectomax” is a new generation of macrolide anti-parasitic drugs developed by Pfizer Inc. in the 1990s. It is composed of mutated avermectin. The Streptomyces strain is biosynthesized in an environment containing cyclohexanoic acid (CHC). Doramectin belongs to avermectins, but has better pharmacokinetic properties and efficacy than avermectin. It has a wide range of antiparasitic, high blood concentration in the body, and slow elimination (duracilin).
  • Doramectin has been approved by the US Food and Drug Administration (FDA) as a treatment for parasitic infections such as gastrointestinal tract nematodes, scorpion worms, ocular parasites, ticks, sucking lice, cockroaches, etc. in cattle, sheep, pigs, etc.
  • FDA US Food and Drug Administration
  • the veterinarian uses an anthelmintic.
  • Dorsalin and avermectin have the same deworming mechanism, which is achieved by strengthening the action of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter that inhibits synaptic posts in the brain.
  • GABA gamma-aminobutyric acid
  • Nerve conduction increased GABA release, increases the normal resting potential of postsynaptic cells, and it is difficult for nerves to transmit stimulation to the muscles, so that the muscles cannot contract, causing parasites to be paralyzed and expelled.
  • Musca domestica is 600-700 times more sensitive to avermectin insecticides and has a high selectivity. It is safer at normal dosages.
  • doramectin batch fermentation process In the current doramectin batch fermentation process, most of them only take pH, DO, temperature, etc. as control The basis and means of the strategy. Most of these parameters can only reflect the performance or operating conditions of the equipment, and can not reflect the physiological state of the larvae of the larvae in real time.
  • the doramectin fermentation process is an aerobic fermentation, especially the early stage of oxygen consumption control has an important impact on the growth and product formation of the production strain.
  • appropriate agitation and aeration must be provided to allow the cells to multiply and accumulate the desired metabolites.
  • the oxygen demand of the cells in different fermentation stages is different, and the oxygen supply capacity of the fermentation liquid directly affects the enzyme activity, metabolic pathway and product yield of the microorganism. Therefore, it is important to study the influence of oxygen supply on fermentation and control to improve production efficiency and improve product quality.
  • the general aerobic fermentation process controls higher oxygen supply to avoid oxygen limitation.
  • the dissolved oxygen concentration (Dissolved Oxygen, DO) can be used to characterize the oxygen supply level by controlling the stirring speed and air flow. Can effectively control the DO.
  • the dissolved oxygen level is affected by both the oxygen supply rate (OTR) and the oxygen consumption rate (OUR).
  • OTR oxygen supply rate
  • OUR oxygen consumption rate
  • the influencing factors of OTR are mainly the rheological properties and operating parameters of the fermentation broth, such as flow rate, stirring speed, stirring slurry type and the like.
  • OUR is a typical physiological parameter that reflects the true oxygen consumption of M. serrata.
  • fermentation generally controls the level of dissolved oxygen above the critical oxygen. Otherwise, the oxygen supply will become a limiting factor, and OUR will change with the change of OTR, which is generally not conducive to fermentation.
  • the technical problem to be solved by the invention is to provide a method for fermentative production of doramectin based on the oxygen consumption rate OUR as a control parameter, adopting a two-stage OUR control strategy, and the high OUR in the early stage enables the hyphae to grow rapidly and quickly produce effects.
  • the doramectin fermentation production method based on the oxygen consumption rate OUR is a control parameter, including the doramectin fermentation process, and the oxygen consumption rate OUR of the cells is calculated in real time during the fermentation process, and the stirring speed and ventilation are provided in the early 0-50 hours.
  • F in is the inlet flow rate in mmol/L/hr; V is the volume of the fermentation broth; O 2in , O 2out is the molar concentration of oxygen in the intake and exhaust gases; N 2in , N 2out is the molar concentration of nitrogen in the intake and exhaust gases .
  • the oxygen consumption rate OUR calculation is performed by using the Yongyu Hengping tail gas mass spectrometer to perform on-line online collection and analysis of the intake and exhaust gases.
  • the ammonium sulfate supplement acceleration rate is 0.038 g ⁇ 0.006 g/lh.
  • the present invention has the following significant advantages:
  • the invention determines an optimized Doppler strain for controlling the control process of the doramectin fermentation process by real-time online analysis of the exhaust gas by the gas mass spectrometer during the fermentation of the doramectin and the online calculation of the software package. The key parameters of the fermentation control process.
  • the higher OUR in the early stage allows the hyphae to grow rapidly and quickly produce potency, and the later control of reasonable OUR levels, reducing the consumption of sugar and hyphae, and promoting the rapid increase of doramectin synthesis, and Maintain a long-term rapid synthesis rate, 260 hours of Doramella
  • the fermentation yield of the prime is as high as 1934 ug/ml.
  • Figure 1 is a graph showing the OUR variation at different oxygen supply levels in the previous period.
  • Figure 2 is a graph showing product synthesis changes at different pre-oxygen consumption rate levels.
  • Figure 3 is a diagram showing the morphology of the cells after 96 hours of differentiation under the control of OUR-A.
  • Figure 4 is a diagram showing the morphology of the cells after 96 hours of differentiation under the control of OUR-B.
  • Figure 5 is a diagram showing the morphology of the cells after 96 hours of differentiation under the control of OUR-C.
  • Fig. 6 is a graph showing different OUR changes in the early stage of OUR-B.
  • Figure 7 is a graph showing the product synthesis changes at different oxygen consumption rate levels in the early stage of OUR-B.
  • Doramectin fermentation uses tertiary fermentation, which includes secondary seed fermentation.
  • the deposited spores were uniformly spread on a slant/plate medium with an inoculation needle or a sterile bamboo stick, and cultured at 28 ° C and 40% relative humidity for 5-7 days.
  • 100 ml of seed medium was added to a 500 ml shake flask, and after sterilization, 3-4 ml of the spore suspension was added and cultured at 28 ° C, 240 rpm for 24-48 hours.
  • Biomass determination Off-line measurement Using the volume method, 10 mL of the fermentation broth was placed in a centrifuge tube, centrifuged at 3000 rpm for 15 min, and the centrifuged supernatant was poured into a graduated cylinder, and the volume of the fermentation broth was calculated from the volume of the supernatant.
  • On-line determination of pH and DO On-line measurement was performed using a Mettler Toledo high temperature electrode.
  • Platinum temperature electrode was measured on-line.
  • Determination of oxygen and nitrogen in the intake and exhaust gases real-time online collection and analysis of the intake and exhaust gases during the fermentation process using the Hanyu Hengping tail gas mass spectrometer.
  • Oxygen consumption rate OUR measurement The calculation of OUR is calculated by analyzing the data of the fermentation tail gas. The equilibrium equation is established by keeping the inert gas N 2 in the intake and exhaust gases constant, and the formula for calculating OUR is as follows:
  • F in is the inlet flow rate in mmol/L/hr; V is the volume of the fermentation broth in kg; O 2in , O 2out is the molar concentration of oxygen in the intake and exhaust; N 2in , N 2out is in the intake and exhaust Nitrogen molar concentration.
  • the doramectin fermentation was carried out in a 50 L fermentor, and the effect of the oxygen consumption rate OUR level and the product synthesis rate of the cells was studied by adjusting the oxygen supply level.
  • the different oxygen supply levels were controlled by adjusting the aeration flow rate and the agitation speed.
  • the metabolic status of the produced bacteria at different oxygen consumption rates was studied, and the whole process was supplemented. Glucose to maintain a certain residual sugar concentration, so that the entire fermentation process is in a non-sugar restricted state.
  • the oxygen supply level of 0-hour hours before the fermentation of doramectin was regulated by the adjustment of stirring speed and aeration, and the oxygen consumption rate OUR was controlled at 38-41mmol/l/h (OUR-A) and 28-30mmol respectively. /l/h(OUR-B) and 18-22 mmol/l/h (OUR-C).
  • OUR-A 38-41mmol/l/h
  • OUR-B 18-22 mmol/l/h
  • the maximum bacterial concentration (PMV 56%) sugar consumption rate was 0.42g / l / h; when the OUR peak control 28-30mmol / l / h, although the initial titer is only 22ug / ml, but the final titer is 1583IU / ml
  • the specific growth rate of 0-50h is 0.0167h -1
  • the maximum bacterial concentration (PMV 52%) is 0.39g/l/h; when the OUR peak is controlled at a low level of 18-22mmol/l/h,
  • the initial control price and the final titer are both lower than the above two control strategies.
  • the specific growth rate in the early stage is only 0.0104h -1 , and the maximum bacterial concentration (PMV 46%) is 0.28g/lh, and the fermentation product synthesis rate is Significantly lower than the other two oxygen consumption rate control conditions, the final fermentation product is only 840ug / ml, possibly due to changes in metabolism.
  • controlling the pre-fermentation OUR to a certain range may be beneficial to the distribution of metabolic flux, especially primary metabolism and secondary metabolism distribution have an important impact on fermentation.
  • the oxygen consumption rate is controlled at 28-30 mmol/l/h (OUR-B)
  • the corresponding hyphae after 96 hours are robust, the hyphae are longer, and the staining is deep.
  • the product synthesis rate is faster (see Figure 4), but at a low oxygen consumption rate of 18-22mmol/l/h (OUR-C), 96 hours of hyphal differentiation is later, while hyphae are finer and lightly stained (see Figure 5).
  • the corresponding product synthesis rate is also significantly lower than the high oxygen consumption rate control batch.
  • the oxygen consumption rate in the early stage was controlled at 28-30mmol/l/h (OUR-B), and the respiratory metabolic activity of the cells was controlled at 25-28mmol by the supplemental flow rate of the available nitrogen source.

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Abstract

提供了一种基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法。所述发酵生产方法采用两段OUR控制策略，多拉菌素发酵过程前期0-50小时通过搅动转速和通气供氧调整控制菌体的氧消耗速率OUR在28-30mmol/l/h，之后50-260小时分化发酵阶段通过搅拌转速、通气供氧及硫酸铵连续补加控制OUR在18-21 mmol/l/h。

Description

基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法 技术领域
本发明涉及多拉菌素生产领域，具体涉及基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法。
背景技术
多拉菌素，商品名为“通灭”(Dectomax)，是20世纪90年代由美国辉瑞公司研制开发的新一代大环内酯类广谱抗寄生虫药，它由突变的阿维菌素链霉菌株在含有环己甲酸(CHC)的环境下生物合成。多拉菌素属于阿维菌素类药物，但具有比阿维菌素更佳的药代动力学特性和功效，其抗寄生虫范围广泛、体内血药浓度高、消除慢(多拉菌素半衰期5.7天，阿维菌素半衰期4.2天)，药效维持时间长、无过敏反应等。多拉菌素已由美国食品药品管理局(FDA)批准作为治疗牛、羊、猪等家畜胃肠道线虫、彪肺虫、眼部寄生虫、蛴螬、吸虱、疥癣螨等寄生虫感染的兽用驱虫药。
多拉菌素与阿维菌素的驱虫机制相同，都是通过加强γ-氨基丁酸(GABA)的作用来实现，GABA是一种抑制性神经递质，在大脑GABA主要抑制突触后神经传导，GABA释放量增加，使突触后细胞的正常休止电位提高，神经难以将刺激传递给肌肉，使肌肉不能收缩，导致寄生虫麻痹而被驱除。家蝇与哺乳动物相比，其对阿维菌素类杀虫剂的敏感程度增加600～700倍，且具有很高的选择性，在正常使用剂量下，安全性较好。
在现行的多拉菌素批培养发酵工艺中，大多仅仅采pH、DO、温度等作为控 制策略的依据和手段。这些参数大多数情况仅仅能反应出设备的性能或者操作条件，而不能实时的、真实的反应出多拉链霉菌的生理状态。多拉菌素发酵过程属于耗氧发酵，尤其是前期的耗氧水平控制对生产菌株的生长和产物形成有着重要的影响。在多拉菌素发酵过程中，必须提供合适的搅拌和通气，菌体才能繁殖和积累所需代谢产物。此外，在不同发酵阶段的菌体的需氧量是不同的，发酵液的供氧能力的大小直接影响微生物的酶的活性、代谢途径及产物产量。因此研究供氧大小对发酵的影响及控制对提高生产效率，改善产品质量等都有重要意义。
一般的耗氧发酵过程均控制较高的供氧以避免氧限制的发生，在这种情况下可以以溶解氧浓度(Dissolved Oxygen,简称DO)来表征供氧水平，通过控制搅拌转速和空气流量可以有效的控制DO。溶氧水平受供氧速率(OTR)和耗氧速率(OUR)两方面影响。其中OTR的影响因素主要有发酵液的流变特性和操作参数，例如流量、搅拌转速、搅拌浆型等。与DO和OTR不同的是，OUR是一个典型的生理参数，能反应出多拉链霉菌真实的耗氧情况。通常来说，发酵一般会将溶氧水平控制在临界氧以上，否则，氧供应就会成为限制性因素，OUR就会随着OTR的变化而变化，这种情况一般是不利于发酵进行的。
发明内容
本发明要解决的技术问题是提供一种基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法，采用两段OUR控制策略，前期用较高的OUR使得菌丝快速生长并快速产生效价，后期控制合理的OUR水平，减少耗糖和菌丝老化，促进多拉菌素合成的快速增加，并维持长时间的快速合成速率，260小时的多拉菌素发酵产量高达1934ug/ml。
本发明通过以下技术方案实现：
基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法，包括多拉菌素发酵过程，在发酵过程中实时计算菌体的耗氧速率OUR，前期0-50小时通过搅拌转速和通气供氧调整控制菌体的耗氧速率OUR值为28-30mmol/l/h，之后50-260小时分化发酵阶段通过搅拌转速、通气供氧及硫酸铵连续补加控制菌体的耗氧速率OUR值为18-21mmol/l/h；
Figure PCTCN2015094999-appb-000001
其中Fin为进气流量，单位mmol/L/hr；V为发酵液体积；O2in，O2out为进气和尾气中氧气摩尔浓度；N2in，N2out为进气和尾气中氮气摩尔浓度。
本发明进一步改进方案是，耗氧速率OUR计算采用舜宇恒平尾气质谱仪对进气和尾气进行实时在线采集分析。
本发明进一步改进方案是，硫酸铵补加速率为0.038g±0.006g/lh。
本发明与现有技术相比，具有以下明显优点：
在现行的多拉菌素批培养发酵工艺中，大多仅仅采pH、DO、温度等作为控制策略的依据和手段。这些参数大多数情况仅仅能反应出设备的性能或者操作条件，而不能实时的、真实的反应出多拉链霉菌的生理状态。本发明通过在多拉菌素发酵过程中气体质谱仪对尾气的实时在线分析以及软件包的在线计算，确定了一种用于指导多拉菌素发酵过程控制方法，得到的最优化多拉菌素发酵控制工艺关键参数。采用两段OUR控制策略，前期用较高的OUR使得菌丝快速生长并快速产生效价，后期控制合理的OUR水平，减少耗糖和菌丝老化，促进多拉菌素合成的快速增加，并维持长时间的快速合成速率，260小时的多拉菌 素发酵产量高达1934ug/ml。
附图说明
图1为前期不同供氧水平下OUR变化曲线图。
图2为不同前期氧消耗速率水平下的产物合成变化图。
图3为OUR-A控制下分化96小时后菌体形态图。
图4为OUR-B控制下分化96小时后菌体形态图。
图5为OUR-C控制下分化96小时后菌体形态图。
图6为前期OUR-B后期不同OUR变化曲线图。
图7为前期OUR-B后期不同氧消耗速率水平下的产物合成变化图。
具体实施方式
一、材料和方法
1、菌种和培养基
生产菌株：多拉链霉菌JT-01，保存于江苏威凌生化有限公司
斜面/平板种子培养基：
淀粉0.6％，酵母抽提物0.4％，硫酸镁0.05％，磷酸氢二钾0.05％，氯化钠0.05％，硝酸钠0.1％，琼脂粉1.5％；pH 7。
摇瓶种子培养基：
淀粉2％，黄豆饼粉1％，磷酸氢二钾0.2％，氯化钠0.1％，碳酸钙0.2％；pH 7。
发酵培养基：
淀粉15％，黄豆饼粉1.2％，酵母提取物0.7％，磷酸氢二钾0.5％，氯化钠0.06％，10％浓度的环己甲酸钠3％；pH 7。
2、仪器和试剂
仪器：发酵罐上海国强生化装备有限责任公司15L和50L发酵罐；舜宇恒平尾气质谱仪及分析软件；722型紫外可见分光光度计；旋转式摇床。
3、培养方法
多拉菌素发酵采用三级发酵，其中包括二级种子发酵。
将保藏的菌种孢子用接种针或无菌竹签均匀涂在斜面/平板培养基上，在28℃和40％相对湿度条件下培养5-7天。
在500ml摇瓶中加入100ml种子培养基，灭菌后接入3-4ml孢子悬浮液，在28℃，240rpm下培养24-48小时。
在500ml锥形瓶中加入100ml发酵培养基，接入8ml种子液，在28℃，240rpm下培养13天。
4、测定方法：
还原糖测定：DNS方法。
生物量测定：离线测定采用体积法，将10mL发酵液置于离心管，3000rpm离心15min，将离心上清倒入量筒，根据上清的体积计算出发酵液的体积。
效价测定：采用国家药典方法。
pH、DO在线测定：采用Mettler Toledo耐高温电极进行在线测定。
温度：铂温度电极在线测定。
进气和尾气中氧和氮的测定:采用舜宇恒平尾气质谱仪对发酵过程中的进气和尾气进行实时在线采集分析。
耗氧速率OUR测定：OUR的计算通过对发酵尾气的分析数据计算得到。以进气和尾气中惰性气体N2维持恒定建立平衡方程，求得OUR的计算公式如下:
Figure PCTCN2015094999-appb-000002
其中Fin为进气流量，单位mmol/L/hr；V为发酵液体积，单位kg；O2in，O2out为进气和尾气中氧气摩尔浓度；N2in，N2out为进气和尾气中氮气摩尔浓度。
二、结果与讨论
1、发酵前期(0-50小时)耗氧速率控制与多拉菌素的合成
在50L发酵罐中进行多拉菌素发酵，通过调节供氧水平来研究菌体的耗氧速率OUR水平及产物合成速率的影响。本研究首次在多拉菌素的发酵过程中，通过调整通气流量和搅拌转速，来控制不同的供氧水平，研究了生产菌在不同耗氧速率状态下的代谢状况，整个过程中通过补加葡萄糖来维持一定的残糖浓度，使整个发酵过程处于非糖限制状态。
通过搅拌转速和通气量的调整来调控多拉菌素发酵前期0-50小时的供氧水平，分别控制其耗氧速率OUR分别在38-41mmol/l/h(OUR-A)、28-30mmol/l/h(OUR-B)和18-22mmol/l/h(OUR-C)。在比较三种控制策略OUR基本变化规律基础上，取三批发酵的数据进行了比较分析结果见图2、表1。从结果看出，当OUR峰值为38-41mmol/l/h，发酵起始效价为40ug/ml，放罐效价为1395ug/ml，0-50h的比生长速率为0.0189h-1，到达最大菌浓(PMV 56％)糖耗 速率为0.42g/l/h；当OUR峰值控制28-30mmol/l/h时，虽起始效价只有22ug/ml，但最终效价为1583IU/ml,0-50h的比生长速率为0.0167h-1，到达最大菌浓(PMV 52％)糖耗速率为0.39g/l/h；当OUR峰值控制18-22mmol/l/h较低水平时，其起始效价和最终效价都低以上两种控制策略，前期的比生长速率只有0.0104h-1，到达最大菌浓(PMV 46％)糖耗速率为0.28g/lh，发酵产物合成速率明显低于其它两种耗氧速率控制条件，最终发酵产物只有840ug/ml，可能代谢发生变化所致。总之，将前期发酵OUR控制在一定范围(28-30mmol/lh)，将可能有利于代谢流的分配，特别是初级代谢和次级代谢分配对发酵产生重要影响。
表1 发酵前期(0-50)OUR控制策略的多拉菌素发酵糖耗、菌体浓度发酵单位
Figure PCTCN2015094999-appb-000003
从图1中可以看出，供氧水平越高菌体的耗氧速率OUR也越高。但是，在高耗氧速率38-41mmol/l/h(OUR-A)的发酵条件下，进行到40小时以后，氧的消耗速率OUR呈现快速下降的趋势，这主要是与发酵过程中菌体的过度分化有关，这是由于过高的OUR促使了菌丝的过早老化，通过显微观察也证实了这点：前期高供氧发酵到96小时左右(见图3)，菌丝不易被染色而且菌丝呈高度断裂状态，发酵液粘度很低。而在耗氧速率控制在28-30mmol/l/h(OUR-B)情况下，96小时对应的分化后菌丝健壮，菌丝较长、染色深，这时的产物合成速率较快(见图4)，但是在低耗氧速率在18-22mmol/l/h(OUR-C)控制条件下，96小时菌丝的分化较晚，同时菌丝较细，染色浅(见图5)，对应的产物合成速率也明显低于高的耗氧速率控制批次。
2、多拉菌素后期(50-260小时)分化后耗氧速率控制与多拉菌素的合成
多拉菌素发酵过程中，后期耗氧速率的维持对于产物合成有着重要影响，尤其是在合成的能量供应和菌丝的分化速率控制方面起着关键作用。因此本研究考察了发酵培养后期分化阶段通过硫酸铵流加控制不同的耗氧速率水平对多拉菌素发酵合成的影响。
根据前面的考察实验结果，将前期的耗氧速率控制在28-30mmol/l/h(OUR-B)，后期通过速效氮源的补加流速调节控制菌体的呼吸代谢活力分别在25-28mmol/l/h(OUR-E)、18-21mmol/l/h(OUR-F)和10-14mmol/l/h(OUR-G)三个水平,OUR变化曲线见图6，多拉菌素发酵合成见附图7及表2。
表2 后期不同耗氧速率情况下的多拉菌素发酵代谢
Figure PCTCN2015094999-appb-000004
在多拉菌素发酵前期通过转速和通气量的调整，控制耗氧速率在28-30mmol/l/h，三批的生长和代谢比较一致，50小时后进入分化期，随着菌丝体的分化断裂，氧的消耗速率显著下降，为了稳定合理的耗氧速率OUR，我们经过大量的研究分析，发现通过一定补加速率硫酸铵补加能够很好地促进耗氧速率的维持。从产物的合成过程变化图(图7)中也能够看出，当合成阶段控制耗氧速率在25-28mmol/l/h时，发酵180小时前的合成速率较高，但之后的合成速率逐渐降低，260小时的发酵产量为1740ug/ml，耗氧速率在18-21mmol/l/h状态下，产物的合成速率维持的最好，260小时的多拉菌素发酵产量最高达到了1934ug/ml。当后期维持较低的耗氧速率水平10-14mmol/l/h时，多拉菌素的合成明显受到限制，最终单位只有1495ug/ml。
基于以上的事实，我们经多拉菌素新工艺采用两段OUR控制策略，前期用较高的OUR(28-30mmol/l/h)使得菌丝快速生长并快速产生效价，后期控制合理的OUR(18-21mmol/l/h)水平，减少耗糖和菌丝老化，促进多拉菌素合成的快速增加，并维持长时间的快速合成速率。

Claims (3)

  1. 基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法，包括多拉菌素发酵过程，其特征在于：在发酵过程中实时计算菌体的耗氧速率OUR，前期0-50小时通过搅拌转速和通气供氧调整控制菌体的耗氧速率OUR值为28-30mmol/l/h，之后50-260小时分化发酵阶段通过搅拌转速、通气供氧及硫酸铵连续补加控制菌体的耗氧速率OUR值为18-21 mmol/l/h；
    Figure PCTCN2015094999-appb-100001
    其中Fin为进气流量，单位mmol/L/hr；V为发酵液体积；O2in，O2out为进气和尾气中氧气摩尔浓度；N2in，N2out为进气和尾气中氮气摩尔浓度。
  2. 根据权利要求1所述的基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法，其特征在于：耗氧速率OUR计算采用舜宇恒平尾气质谱仪对进气和尾气进行实时在线采集分析。
  3. 根据权利要求1所述的基于耗氧速率OUR为控制参数的多拉菌素发酵生产方法，其特征在于：硫酸铵补加速率为0.038g±0.006g/lh。
PCT/CN2015/094999 2015-09-22 2015-11-19 基于耗氧速率our为控制参数的多拉菌素发酵生产方法 WO2017049751A1 (zh)

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