CN110511974A - 一种提高红霉素发酵效价的发酵方法 - Google Patents
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- C12P19/62—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin the hetero ring having eight or more ring members and only oxygen as ring hetero atoms, e.g. erythromycin, spiramycin, nystatin
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Abstract
本发明涉及一种提高红霉素发酵效价的发酵方法,该发酵方法是采用以红色链霉菌为发酵菌种、由种子培养和发酵培养构成的二级发酵模式发酵生产,本发明通过改变种子及发酵培养基的配方结构,祛除了黄豆饼粉的使用,补入硝酸钠溶液作为氮源,并引入微量元素钴,改善了发酵过程中的供氧条件,降低了供氧的能源消耗,从而增强菌体活力,延长红霉素分泌期,提高了红霉素发酵水平。
Description
技术领域
本发明涉及微生物发酵领域,特别是涉及一种提高红霉素发酵效价的发酵方法。
背景技术
红霉素(erythromycin,Er)是一种碱性抗生素,主要包括红霉素A(ErA)、红霉素B(ErB)、红霉素C(ErC)和红霉素E(ErE)等组分,其抗菌谱与青霉素近似,临床主要应用于链球菌引起的扁桃体炎、猩红热、白喉及带菌者、淋病等。近年来随着对红霉素的研究开发,使红霉素的需求量持续增加。
现有技术中,红霉素的生产多采用三级发酵模式,其培养基中都含有大量的迟效氮源,如黄豆饼粉等。黄豆饼粉等迟效有机氮源的使用,增加了发酵液的粘度,影响了供氧,造成发酵液氧传递率低,导致在发酵过程中供氧不足影响菌种生长状况,进而降低了红霉素产素能力。同时菌种对培养基中的迟效氮源消耗慢,发酵结束残留较大,影响提取的效果及成品质量;并且发酵液放罐时大量的残留给后续提取造成困难。
发明内容
本发明的目的就在于克服上述现有技术的缺陷,提供一种有效提高红霉素产素能力及成品品质的红霉素发酵方法。
为实现上述发明目的所采取的技术方案为:
一种提高红霉素发酵效价的发酵方法,其特征在于采用以红色链霉菌为发酵菌种、由种子培养和发酵培养构成的二级发酵模式,
其中种子培养所用的种子培养基组成为:淀粉2~4%,豆粉2~4%,糊精1~3%,玉米浆1~3%,硫酸铵0.1~0.3%,轻质碳酸钙0.3~0.8%,硫酸镁0.1~0.3%,氯化钴0.01~0.03%,适量二甲基硅油消沫剂,其余为水,以重量百分比计;
发酵培养所用的发酵培养基组成为:淀粉2.0~4.0%,玉米浆2.0~4.0%,硫酸铵0.1~0.4%,碳酸钙0.7~1.0%,硫酸镁0.1~0.3%,氯化钴0.02~0.05%,适量二甲基硅油消沫剂,其余为水,以重量体积比计。
所述种子培养条件为:培养温度34~36℃,通气量1:08~1.0v/v/min,培养时间45~50hr。
发酵培养条件为:培养温度32~36℃,溶氧大于20%,培养时间160~170hr。
在发酵过程中进行补料,包括补硝酸钠、补糖和补正丁醇,其中
补硝酸钠:在发酵培养40~150hr补入硝酸钠溶液,控制发酵液中氨基氮含量20~30%;
补糖:当发酵液中总糖降至1.8%以下时开始补入葡萄糖溶液,控制发酵液中总糖1.0~2.0%;
补正丙醇:在发酵20hr后补入正丁醇,控制发酵液中正丙醇含量0 .05~0 .10%。
所述硝酸钠溶液质量体积浓度为15~25%,葡萄糖溶液质量浓度为20~30%,正丙醇溶液质量浓度为30~40%。
本发明在种子培养基和发酵培养基中加入微量元素钴,激活菌种体内各种酶,改变了菌体的代谢能力和途径;在发酵培养基中去除了黄豆饼粉的使用,补入硝酸钠溶液作为氮源,稀化了发酵培养基,改善了发酵过程中的供氧条件,降低了供氧的能源消耗,从而增强菌体活力,延长红霉素分泌期,提高了红霉素发酵水平。本发明通过对种子及发酵培养基的配方结构改变,补料的配方结构及控制方式改变,并引入微量元素钴,从而改变了菌体的代谢途径,由原工艺的三级发酵缩短为二级发酵,提高了发酵效价,缩短了发酵周期,降低了原材料成本。同时由于迟效碳氮源的减少,红霉素的提取更为容易,成品质量也获得提高。
具体实施方式
下面用实例,对本技术方案进行具体的描述,应该理解的是,实例是用于说明本发明而不是对本发明的限制。本发明的范围与核心内容依据权利要求书加以确定。
实施例1
1.种子培养:
种子培养基组成为:淀粉2%,豆粉2%,糊精:2%,玉米浆3%,硫酸铵0.25%,轻质碳酸钙0.70%,硫酸镁0.15%,氯化钴0.02%,适量二甲基硅油消沫剂,其余为水,以重量百分比计。灭菌备用。
将斜面红色链霉菌孢子接种于灭菌好的一级种子培养基中培养,培养温度34℃,通气量1:0 .8v/v/min,搅拌,培养45hr得种子液。
2.发酵培养:
发酵培养基组成为:淀粉3.0%,玉米浆3.0%,硫酸铵0.3%,碳酸钙0.80%,硫酸镁0.2%,氯化钴0.03%,适量二甲基硅油消沫剂,其余为水,以重量体积比计。
将过程1所得的种子液按20%接种量接种于灭菌好的发酵培养基中培养,培养温度32~36℃,通气,搅拌,控制发酵过程溶氧20%以上,培养过程中监测氨基氮和总糖:20hr开始补入正丙醇,其浓度40%,补入量控制发酵液中丙醇含量0 .05~0 .10%为宜。在40小时补入硝酸钠溶液,控制发酵液中氨基氮含量20~30%;总糖降至1 .8%以下时开始补糖,控制总糖1 .0~2.0%。培养至160~170hr时停止发酵,得含有红霉素的发酵液。放罐效价9296μg/ml 。
实施例2
1.种子培养:
种子培养基组成为:淀粉3%,豆粉3%,糊精:3%,玉米浆4%,硫酸铵0.20%,轻质碳酸钙0.8%,硫酸镁0.20%,氯化钴0.04%,适量二甲基硅油消沫剂,其余为水,以重量百分比计。灭菌备用。
将斜面红色链霉菌孢子接种于灭菌好的一级种子培养基中培养,培养温度34℃,通气量1:0 .8v/v/min,搅拌,培养45hr得种子液。
2 .发酵培养:
发酵培养基组成为:淀粉4.0%,玉米浆4.0%,硫酸铵0.2%,碳酸钙0.65%,硫酸镁0.3%,氯化钴0.04%,适量二甲基硅油消沫剂,其余为水,以重量体积比计。
将过程1所得的种子液按20%接种量接种于灭菌好的发酵培养基中培养,培养温度32~36℃,通气,搅拌,控制发酵过程溶氧20%以上,培养过程中监测氨基氮和总糖:20hr开始补入正丙醇,其浓度40%,补入量控制发酵液中丙醇含量0 .05~0 .10%为宜。在40小时补入硝酸钠溶液,控制发酵液中氨基氮含量20~30%;总糖降至1 .8%以下时开始补糖,控制总糖1 .0~2.0%。培养至160~170hr时停止发酵,得含有红霉素的发酵液。放罐效价9145μg/ml 。
实施例3
1 .种子培养:
种子培养基组成为:淀粉4%,豆粉3%,糊精:4%,玉米浆3%,硫酸铵0.25%,轻质碳酸钙0.6%,硫酸镁0.25%,氯化钴0.03%,适量二甲基硅油消沫剂,其余为水,以重量百分比计。灭菌备用。
将斜面红色链霉菌孢子接种于灭菌好的一级种子培养基中培养,培养温度34℃,通气量1:0 .8v/v/min,搅拌,培养45hr得种子液。
2 .发酵培养:
发酵培养基组成为:淀粉3.0%,玉米浆4.0%,硫酸铵0.3%,碳酸钙0.60%,硫酸镁0.3%,氯化钴0.05%,适量二甲基硅油消沫剂,其余为水,以重量体积比计。
将过程1所得的种子液按20%接种量接种于灭菌好的发酵培养基中培养,培养温度32~36℃,通气,搅拌,控制发酵过程溶氧20%以上,培养过程中监测氨基氮和总糖:20hr开始补入正丙醇,其浓度40%,补入量控制发酵液中丙醇含量0 .05~0 .10%为宜。在40小时补入硝酸钠溶液,控制发酵液中氨基氮含量20~30%;总糖降至1 .8%以下时开始补糖,控制总糖1 .0~2.0%。培养至160~170hr时停止发酵,得含有红霉素的发酵液。放罐效价8932μg/ml 。
对比实施例
1 .一级种子培养:
将斜面孢子接种于灭菌好的一级种子培养基中培养,培养温度34~36℃,通气量1:1.2~1 .5v/v/min,搅拌,培养50~60hr得一级种子液。
一级种子培养基组成:淀粉2%,糊精1 .5%,黄豆饼粉3.%,硫酸铵0 .4%,玉米浆0.6%,氯化钠0 .4%,碳酸钙1 .5%,豆油0 .20%,其余为水;在122~124℃,保压30分钟。
2 .二级种子培养:
将一级种子液按接种量15%接种于灭菌好的二级种子培养基中培养,培养温度34~35℃,通气量1:1.2~1.5v/v/min,搅拌,培养26~28hr得二级种子液。
二级种子培养基组成:淀粉3 .5%,糊精1 .5%,黄豆饼粉3 .0%,硫酸铵0 .2%,玉米浆0 .8%,氯化钠0 .2%,碳酸钙0 .6%,豆油0 .2%,其余为水;在122~124℃,保压30分钟。
3 .发酵培养:
发酵培养基组成:淀粉4 .0%,糊精3.0%,黄豆饼粉3.0%,硫酸铵0 .2%,玉米浆0 .4%,氯化钠0 .3%,碳酸钙0 .6%,豆油0 .2%,其余为水;在122~124℃,保压30分钟。
过程补料:
葡萄糖40%,发酵过程中需要补入发酵液体积的30~40% ,流加补入,控制pH6.9~7.1。
正丙醇浓度40%,其补入量以始终控制发酵液中丙醇含量0 .03~0 .06%为宜。
植物油的补入量以控制发酵液中残油0 .05~0 .01%为宜。
将二级种子液按20%接种量接种于灭菌好的发酵培养基中培养,培养温度32~34℃,通气量1:1 .1~1 .4v/v/min,搅拌,发酵过程溶氧30%以上,培养20hr开始补入糖、植物油、正丙醇,经160~170hr培养得含有红霉素的发酵液。效价8328μg/ml。
Claims (5)
1.一种提高红霉素发酵效价的发酵方法,其特征在于采用以红色链霉菌为发酵菌种、由种子培养和发酵培养构成的二级发酵模式发酵生产,
其中种子培养所用的种子培养基组成为:淀粉2~4%,豆粉2~4%,糊精1~3%,玉米浆1~3%,硫酸铵0.1~0.3%,轻质碳酸钙0.3~0.8%,硫酸镁0.1~0.3%,氯化钴0.01~0.03%,适量二甲基硅油消沫剂,其余为水,以重量百分比计;
发酵培养所用的发酵培养基组成为:淀粉2.0~4.0%,玉米浆2.0~4.0%,硫酸铵0.1~0.4%,碳酸钙0.7~1.0%,硫酸镁0.1~0.3%,氯化钴0.02~0.05%,,适量二甲基硅油消沫剂,其余为水,以重量体积比计。
2.按照权利要求1所述的提高红霉素发酵效价的发酵方法,其特征在于所述种子培养条件为:培养温度34℃~36℃,通气量1:08~1.0v/v/min,培养时间45 hr~50hr。
3.按照权利要求1所述的提高红霉素发酵效价的发酵方法,其特征在于所述发酵培养条件为:培养温度32℃~36℃,溶氧大于20%,培养时间160~170hr。
4.按照权利要求1或3所述的提高红霉素发酵效价的发酵方法,其特征在于,在发酵过程中进行补料,包括补硝酸钠、补糖和补正丁醇,其中
补硝酸钠:在发酵培养40~150hr补入硝酸钠溶液,控制发酵液中氨基氮含量20~30%;
补糖:当发酵液中总糖降至1.8%以下时开始补入葡萄糖溶液,控制发酵液中总糖1.0~2.0%;
补正丙醇:在发酵20hr后补入正丁醇,控制发酵液中正丙醇含量0 .05~0 .10%。
5.按照权利要求4所述的提高红霉素发酵效价的发酵方法,其特征在于所述硝酸钠溶液质量体积浓度为15~25%,葡萄糖溶液质量浓度为20~30%,正丙醇溶液质量浓度为30~40%。
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