Actinomadura yumaensis发酵生产马杜霉素的研究
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摘要
马杜霉素是一种新型的聚醚类离子载体型抗生素，可以特异性的杀灭和抑制对畜牧养殖业危害极大的动物寄生虫——球虫。马杜霉素具有抗球虫谱广，疗效高，用量少，球虫不易产生抗药性等优点，所以被广泛使用。目前有关发酵生产马杜霉素的文献报道极少，研究其发酵生产工艺对于国内马杜霉素的生产具有一定的指导意义。
本文对马杜霉素的测定、利用菌种Actinomadura yumaensis发酵生产马杜霉素的工艺、以及从发酵液中提取马杜霉素的方法进行了研究。
建立了分光光度法测定发酵液中马杜霉素的方法。用甲醇从菌丝体中提取马杜霉素；提取液在高温下与香草醛试剂反应显红色，显色条件为：75℃，20min；显色液冷却后在520nm波长下用分光光度计测定其吸光值，再与标准曲线对照可得马杜霉素的浓度。
在摇瓶条件下对马杜霉素的发酵进行了基础研究，优化后的发酵条件为：种子液菌龄60h；发酵培养基中适宜的碳源为葡萄糖，适宜的氮源为1:1复合使用的豆饼粉+玉米浆，合适的碳氮比为12:1；正交实验优化得到的发酵培养基配方为：葡萄糖7％，豆饼粉1.2％，玉米浆1.2％，NaCl 0.3％，CaCO_3 0.1％，K_2HPO_3 0.15％，Fe_2(SO_4)_3 0.015％；适宜的培养基初始pH6.6，培养温度34℃。
利用3.7L的发酵罐进行马杜霉素发酵实验，结果表明通气量和搅拌转速是影响马杜霉素发酵单位高低的重要因素，适宜的通气量为1.0vvm；搅拌转速以分段控制为佳：在0～60h为300rpm，60～120h为500rpm，120～192h为600rpm，发酵周期为192h。在此条件下，马杜霉素分批发酵的发酵单位达到了8336μg／ml。
研究了3.7L发酵罐条件下补料分批发酵的工艺，发现补料发酵提高了发酵单位，而且连续补料的方式比间歇补料更具有优势。在间歇补料分批发酵工艺中，先采用较低的底物浓度，发酵36h后，分10次间歇补料，最终发酵单位提高到9645μg／ml，比分批不补料发酵提高了15.7％；在连续补料分批发酵工艺中，发酵36h后，以0.0056g葡萄糖／L发酵液／min的速率匀速流加补料，补至发酵156h时，使底物浓度维持在适宜的水平，从而延长了发酵进程中的稳定期，最终发酵单位提高到10218μg／ml，比分批不补料发酵提高了22.6％，比间歇补料分批发酵提高了6％。
初步研究了从发酵液中提取马杜霉素的方法：发酵液经预处理后用过滤或离
浙江大学硕士学位论文
摘要
心的方法收集菌丝体，再在50℃下搅拌12h使其自溶破胞，用乙酸乙酷作为提
取马杜霉素的有机溶剂，在室温下搅拌提取8~12h，提取液再经真空浓缩和低
温结晶得到了马杜霉素的粗晶体。
Maduramicin is a new type of polyether ionophore antibiotic, which can particularly kill and restrain coccidia - a kind of animal parasite which induce coccidiosis. As a widely used anticoccidial drug, maduramicin has a wide anticoccidial spectrum, and it is effective at a low dose rate of 5 mg/kg to prevent the infection of various species of Eimeria.
The quantitative analysis method of maduramicin., the maduramicin production by Actinomadura yumaensis, and the method of extracting maduramicin from broth were studied in this thesis.
Spectrophotometric analysis -method was respectively established for maduramicin quantitative measurement. First extracted maduramicin from the Actinomadura yumaensis mycelium using methanol; then made the maduramicin extracts react with vanilin, the reaction conditions were: 75℃, 20min; finally under 520nm light wavelength, using the spectrophotometer determined the absorbency of the maduramicin extracts, and the maduramicin concentration can be obtained by consulting the standard absorbency-concentration curve.
The maduramicin fermentation was studied using Actinomadura yumanense strain in shaking flask. The results showed that the suitable conditions were: glucose 7 %, soybean powder 1.2%, cornsteep liquitor1.2 %, NaCl 0.3%, CaCO3 0.1%, K2HPO3 0.15 %, Fe2(SO4)3 0.015 %, the initial pH value of the media was 6.6, and the fermentation temperature was 34℃.
The results of batch fermentation in a 3.7L ferrnenter showed that the stirring speed and aeration had obviously influence on the maduramicin production. The suitable aeration rate was 1.0wm, and the stirring speed should be controlled according following subsection: 300rpm in 0-60h period, 500rpm in 60~120h period, 600rpm in 120~192h period, the whole fermentation period was 192h. In these conditions, the maduramicin yield was 8336μg /ml.
Two types of fed-batch processes were studied. Under the intermissive fed-batch process, the initial glucose concentration was 40g/L, and the feeding substrate was added in 10 times after 36h cultivation, the total amount of feeding glucose was 30g/L, finally the maduramicin yield was 9645μg/ml. Under the continuous fed-batch
process, the feeding substrate was added with a even speed of 0,0056g(glucose) /L/min from 36h to 156h, and the maduramicin yield reached 10218μg/ml, which was 22.6% higher than that obtained under the batch process.
A process of extracting maduramicin from fermentation broth was established. After pretreatment, the Actinomadura yumaensis mycelium was obtained by filtration or centrifugation. Under 50℃ let the mycelium autolyze 12h with agitation. By comparison, chose ethyl acetate as the extraction agent; under room temperature, with agitation, extracted the mycelium 8~10h, the maduramicin extraction ratio reached 98%. Finally by vacuum evaporation and crystallization, the maduramicin crystal could be obtained from maduramicin extracts.
本文对马杜霉素的测定、利用菌种Actinomadura yumaensis发酵生产马杜霉素的工艺、以及从发酵液中提取马杜霉素的方法进行了研究。
建立了分光光度法测定发酵液中马杜霉素的方法。用甲醇从菌丝体中提取马杜霉素；提取液在高温下与香草醛试剂反应显红色，显色条件为：75℃，20min；显色液冷却后在520nm波长下用分光光度计测定其吸光值，再与标准曲线对照可得马杜霉素的浓度。
在摇瓶条件下对马杜霉素的发酵进行了基础研究，优化后的发酵条件为：种子液菌龄60h；发酵培养基中适宜的碳源为葡萄糖，适宜的氮源为1:1复合使用的豆饼粉+玉米浆，合适的碳氮比为12:1；正交实验优化得到的发酵培养基配方为：葡萄糖7％，豆饼粉1.2％，玉米浆1.2％，NaCl 0.3％，CaCO_3 0.1％，K_2HPO_3 0.15％，Fe_2(SO_4)_3 0.015％；适宜的培养基初始pH6.6，培养温度34℃。
利用3.7L的发酵罐进行马杜霉素发酵实验，结果表明通气量和搅拌转速是影响马杜霉素发酵单位高低的重要因素，适宜的通气量为1.0vvm；搅拌转速以分段控制为佳：在0～60h为300rpm，60～120h为500rpm，120～192h为600rpm，发酵周期为192h。在此条件下，马杜霉素分批发酵的发酵单位达到了8336μg／ml。
研究了3.7L发酵罐条件下补料分批发酵的工艺，发现补料发酵提高了发酵单位，而且连续补料的方式比间歇补料更具有优势。在间歇补料分批发酵工艺中，先采用较低的底物浓度，发酵36h后，分10次间歇补料，最终发酵单位提高到9645μg／ml，比分批不补料发酵提高了15.7％；在连续补料分批发酵工艺中，发酵36h后，以0.0056g葡萄糖／L发酵液／min的速率匀速流加补料，补至发酵156h时，使底物浓度维持在适宜的水平，从而延长了发酵进程中的稳定期，最终发酵单位提高到10218μg／ml，比分批不补料发酵提高了22.6％，比间歇补料分批发酵提高了6％。
初步研究了从发酵液中提取马杜霉素的方法：发酵液经预处理后用过滤或离
浙江大学硕士学位论文
摘要
心的方法收集菌丝体，再在50℃下搅拌12h使其自溶破胞，用乙酸乙酷作为提
取马杜霉素的有机溶剂，在室温下搅拌提取8~12h，提取液再经真空浓缩和低
温结晶得到了马杜霉素的粗晶体。
Maduramicin is a new type of polyether ionophore antibiotic, which can particularly kill and restrain coccidia - a kind of animal parasite which induce coccidiosis. As a widely used anticoccidial drug, maduramicin has a wide anticoccidial spectrum, and it is effective at a low dose rate of 5 mg/kg to prevent the infection of various species of Eimeria.
The quantitative analysis method of maduramicin., the maduramicin production by Actinomadura yumaensis, and the method of extracting maduramicin from broth were studied in this thesis.
Spectrophotometric analysis -method was respectively established for maduramicin quantitative measurement. First extracted maduramicin from the Actinomadura yumaensis mycelium using methanol; then made the maduramicin extracts react with vanilin, the reaction conditions were: 75℃, 20min; finally under 520nm light wavelength, using the spectrophotometer determined the absorbency of the maduramicin extracts, and the maduramicin concentration can be obtained by consulting the standard absorbency-concentration curve.
The maduramicin fermentation was studied using Actinomadura yumanense strain in shaking flask. The results showed that the suitable conditions were: glucose 7 %, soybean powder 1.2%, cornsteep liquitor1.2 %, NaCl 0.3%, CaCO3 0.1%, K2HPO3 0.15 %, Fe2(SO4)3 0.015 %, the initial pH value of the media was 6.6, and the fermentation temperature was 34℃.
The results of batch fermentation in a 3.7L ferrnenter showed that the stirring speed and aeration had obviously influence on the maduramicin production. The suitable aeration rate was 1.0wm, and the stirring speed should be controlled according following subsection: 300rpm in 0-60h period, 500rpm in 60~120h period, 600rpm in 120~192h period, the whole fermentation period was 192h. In these conditions, the maduramicin yield was 8336μg /ml.
Two types of fed-batch processes were studied. Under the intermissive fed-batch process, the initial glucose concentration was 40g/L, and the feeding substrate was added in 10 times after 36h cultivation, the total amount of feeding glucose was 30g/L, finally the maduramicin yield was 9645μg/ml. Under the continuous fed-batch
process, the feeding substrate was added with a even speed of 0,0056g(glucose) /L/min from 36h to 156h, and the maduramicin yield reached 10218μg/ml, which was 22.6% higher than that obtained under the batch process.
A process of extracting maduramicin from fermentation broth was established. After pretreatment, the Actinomadura yumaensis mycelium was obtained by filtration or centrifugation. Under 50℃ let the mycelium autolyze 12h with agitation. By comparison, chose ethyl acetate as the extraction agent; under room temperature, with agitation, extracted the mycelium 8~10h, the maduramicin extraction ratio reached 98%. Finally by vacuum evaporation and crystallization, the maduramicin crystal could be obtained from maduramicin extracts.
引文
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