CN112080438A - 一种灰黄霉素低产沫菌种及低产沫菌种的制备方法和应用 - Google Patents
一种灰黄霉素低产沫菌种及低产沫菌种的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种灰黄霉素低产沫菌种及低产沫菌种的制备方法和应用,该灰黄霉素低产沫菌种为展青霉(Penicillium patulum)GLINT 19‑21‑30‑2#1,保藏编号为CGMCC NO.20257。本发明的灰黄霉素低产沫菌种用于发酵生产灰黄霉素。本发明制备出的灰黄霉素低产沫菌株能够降低生产成本和能耗,降低发酵液涌泡造成染菌的几率和生产废液的COD,避免使用消泡剂造成的其他异常情况,提高灰黄霉素的产量和质量。本发明最终制备出灰黄霉素低产沫菌株,发酵培养过程中产沫量显著减少,发酵过程不再添加消泡剂,缩减操作步骤,降低能耗,进一步降低了生产成本,同时提高了菌株的单位效价并提高了收率。
Description
技术领域
本发明属于生物制药技术领域,涉及微生物菌种的选育,具体的说是利用灰黄霉素产生菌-展青霉菌为出发菌株制备得到了一种灰黄霉素低产沫菌种。
背景技术
灰黄霉素为抗生素类原料药,中文别名灰霉素或微晶灰黄霉素,灰黄霉素成品为白色或类白色的微细粉末,其主要针对毛发癣菌、小孢子菌、表皮癣菌等浅表部真菌具有良好的抗菌作用,灰黄霉素适用于各种癣病的治疗,包括头癣、须癣、体癣、股癣、足癣和甲癣。现有发酵技术中的灰黄霉素菌种不够优良,发酵培养过程中产沫量较大,为了消除泡沫必须辅加设置消泡剂贮存、计量、输送、滴加和消毒系统,使得生产成本高、耗能高、劳动生产率低,同时也增加了染菌风险。消泡剂的加入会提高生产废液的化学需氧量(COD),最主要的是消泡剂由于自身性质对菌丝代谢产生抑制作用,影响发酵生产能力,最终影响灰黄霉素成品原料药的品质和产量,因此选育制备出一种低产沫菌种用于发酵生产十分必要。
发明内容
本发明为了克服现有技术存在的不足,提供一种灰黄霉素低产沫菌种及低产沫菌种的制备方法和应用。
本发明是通过以下技术方案实现的:本发明公开了一种灰黄霉素低产沫菌种,该灰黄霉素低产沫菌种为展青霉(Penicillium patulum)GLINT 19-21-30-2#1,保藏编号为CGMCC NO.20257。
本发明还公开了一种灰黄霉素低产沫菌种的制备方法,该制备方法具体包括如下步骤:
(1)用消好菌的接种针从砂土管内挑出少量展青霉菌4541的砂土孢子,反复、均匀地涂布在斜面培养基上,将接好种的斜面置于温度为28±1℃培养箱内培养7~8天;
(2)培养成熟后,挑选出外观为白色、孢子丰满、疏密均匀、边缘整齐、背面为红棕色的斜面,用消好菌的无菌玻璃珠将孢子打下,加入消好菌的纯化水20ml,再次充分振摇玻璃珠5分钟;
(3)取装有滤纸和药棉的玻璃漏斗过滤,过滤后取滤液,从中吸取2.0ml于消好菌的空白平碟中稀释作对照,另取滤液8~10ml于双碟中,用紫外线均匀照射15~20分钟,照毕,盖上双碟,用黑布罩住,并关闭净化台日光灯,借助缓冲间的灯光,以极快的速度稀释成浓度为10-5cfu/ml和10-6cfu/ml的稀释液,然后分别吸取浓度为10-5cfu/ml和10-6cfu/ml的稀释液于事先配制好的含有LiCl的分离培养基平板中,每皿0.1ml菌液,将同一浓度的平板用玻璃耙子耙匀分散后,移入28±1℃温度条件下恒温室培养7~8天;
(4)培养成熟后,分生孢子单菌落外观为大小均匀、丰富、厚实,孢子雪白且结合紧密,用接种针挑取分生孢子单菌落移入斜面试管,斜面培养基的配方与分离培养基配方相同,并注明剂量和编号,移至28±1℃温度条件下恒温室培养;
(5)培养7~8天后,斜面雪白一片、表面孢子结合紧密、背部色素为浅色,将培养成熟的菌落接入摇瓶中,并于30±1℃温度条件下恒温振荡12~13天,取下摇瓶测定体积并进行含量分析,选取体积小、效价单位高于初始菌种3%的15~20个斜面,转接斜面并编号;
(6)用已选取编号的斜面,通过挖块进行摇瓶复筛,培养和接种工艺同上一步骤;
(7)培养基不变,初筛后还需经过2-3次复筛培养,才能够得到灰黄霉素低产沫菌种CGMCC NO.20257。
本发明灰黄霉素低产沫菌种的制备方法还包括砂土管的制备步骤:取黄沙和黄土适量,黄沙和黄土用盐酸溶液浸泡24小时后倒出,用纯化水冲洗至pH=7,烘干;将经过处理的黄沙和黄土以1:1的体积比混合均匀,装入10ml试管中,每支装量1克,并塞上棉塞,用牛皮纸包好;砂土管经高压灭菌间歇5-6次,每次时间为1小时,消毒温度121~128℃,0.10~0.15Mpa,检测无菌则试验合格。
本发明灰黄霉素低产沫菌种的制备方法还包括留种保藏步骤:选取复筛后体积小、效价单位高的5~6个对应斜面孢子,用接种刀挑取一小块生长丰满、外观正常的斜面菌落置于三角瓶内,用吸管吸取灭菌水少量于三角瓶内,充分震荡,洗下孢子,制成悬浮液;将悬浮液接入砂土管内,每支砂土管接入0.2~0.3ml悬浮液,塞上棉塞,包上小纱布,绳子扎紧;放入真空干燥箱内,抽真空,取出砂土管移入冰箱,2~8℃保存。
本发明还公开了一种灰黄霉素低产沫菌种CGMCC NO.20257的应用,该灰黄霉素低产沫菌种CGMCC NO.20257用于发酵生产灰黄霉素。
原始菌种最早来源于原上海新华联制药,吴松刚灰黄霉素产生菌展青霉菌(Penicillium patulum )4541。现本发明生产出的灰黄霉素低产沫菌种CGMCC NO.20257保存在砂土管中,为展青霉菌培养得到的后代菌种,为申请人独立培养,市面无售卖。生产以来通过自然分离的方法,每年纯化两次,选取高效价的单菌落。目前使用的灰黄霉素低产沫菌种编号为CGMCC NO.20257,本发明选育出的低产沫菌种属于一种新型菌种,申请人对该菌种进行了生物保藏。
本发明制备出的新菌种GLINT 19-21-30-2#1属于展青霉菌(Penicilliumpatulum)4541培养的后代,市面上无法购买。展青霉菌又称荨麻青霉(P.urticae)。真菌门、半知菌亚门、丝孢纲、丝孢目、丝孢科、青霉属中的一种,属于不对称青霉组,束状青霉亚组。主要分布于土壤、腐烂植物体和羊粪等基质上。
本发明的灰黄霉素低产沫菌种CGMCC NO.20257培养后菌落生长较慢,12~14天后,直径约2.0~2.5 cm,表面大多有放射状沟纹,边缘陡峭,中央稍凸,表面呈粒状,有些在边缘有明显的菌丝束,有的则呈絮状、厚密,灰绿色至亮灰色;有些菌株产生近无色的渗出液;菌落反面暗黄色、橙褐色至红褐色,稍扩散入培养基中。帚状枝疏松散开,有3~4层分枝,其大小和构造差别大,一般40~50μm(极限为20~80μm)。分生孢子链略散,长达50~100μm。分生孢子梗一部分单生,一部分集结成束,多弯曲,梗壁光滑,一般400~520μm×3~4μm。副枝散开,大多15~20μm×3.0~3.5μm。梗基较短,大多7~9μm×3.0~3.5μm。小梗短,4.5~6.5μm×2.0~2.5μm,8~10个密集成一簇。分生孢子椭圆形,后变为近球形,长轴2.5~3.0μm,表面光滑,代谢产生灰黄霉素。
本发明的有益效果是:本发明的目的在于制备出灰黄霉素低产沫菌株,降低生产成本和能耗,降低发酵液涌泡造成染菌的几率和生产废液的COD,避免使用消泡剂造成的其他异常情况,提高灰黄霉素的产量和质量。本发明最终制备出灰黄霉素低产沫菌株,发酵培养过程中产沫量显著减少,发酵过程不再添加消泡剂,缩减操作步骤,降低能耗,进一步降低了生产成本,同时提高了菌株的单位效价并提高了收率。
微生物保藏说明
本发明中要求保护的灰黄霉素低产沫菌种为展青霉(Penicillium patulum)GLINT19-21-30-2#1。该菌种保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址:北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏日期为2020年9月9日,保藏编号为CGMCC NO.20257。
具体实施方式
以下结合具体实施方式对本发明作详细描述。
本发明公开了一种灰黄霉素低产沫菌种,该灰黄霉素低产沫菌种为展青霉(Penicillium patulum)GLINT 19-21-30-2#1,保藏编号为CGMCC NO.20257。
本发明还公开了一种灰黄霉素低产沫菌种的制备方法,该制备方法具体包括如下步骤:
(1)用消好菌的接种针从砂土管内挑出少量展青霉菌4541的砂土孢子,反复、均匀地涂布在斜面培养基上,将接好种的斜面置于温度为28±1℃培养箱内培养7~8天;
(2)培养成熟后,挑选出外观为白色、孢子丰满、疏密均匀、边缘整齐、背面为红棕色的斜面,用消好菌的无菌玻璃珠将孢子打下,加入消好菌的纯化水20ml,再次充分振摇玻璃珠5分钟;
(3)取装有滤纸和药棉的玻璃漏斗过滤,过滤后取滤液,从中吸取2.0ml于消好菌的空白平碟中稀释作对照,另取滤液8~10ml于双碟中,用紫外线均匀照射15~20分钟,照毕,盖上双碟,用黑布罩住,并关闭净化台日光灯,借助缓冲间的灯光,以极快的速度稀释成浓度为10-5cfu/ml和10-6cfu/ml的稀释液,然后分别吸取浓度为10-5cfu/ml和10-6cfu/ml的稀释液于事先配制好的含有LiCl的分离培养基平板中,每皿0.1ml菌液,将同一浓度的平板用玻璃耙子耙匀分散后,移入28±1℃温度条件下恒温室培养7~8天;
(4)培养成熟后,分生孢子单菌落外观为大小均匀、丰富、厚实,孢子雪白且结合紧密,用接种针挑取分生孢子单菌落移入斜面试管,斜面培养基的配方与分离培养基配方相同,并注明剂量和编号,移至28±1℃温度条件下恒温室培养;
(5)培养7~8天后,斜面雪白一片、表面孢子结合紧密、背部色素为浅色,将培养成熟的菌落接入摇瓶中,并于30±1℃温度条件下恒温振荡12~13天,取下摇瓶测定体积并进行含量分析,选取体积小、效价单位高于初始菌种3%的15~20个斜面,转接斜面并编号;
(6)用已选取编号的斜面,通过挖块进行摇瓶复筛,培养和接种工艺同上一步骤;
(7)培养基不变,初筛后还需经过2-3次复筛培养,才能够得到灰黄霉素低产沫菌种CGMCC NO.20257。
本发明灰黄霉素低产沫菌种的制备方法还包括砂土管的制备步骤:取黄沙和黄土适量,黄沙和黄土用盐酸溶液浸泡24小时后倒出,用纯化水冲洗至pH=7,烘干;将经过处理的黄沙和黄土以1:1的体积比混合均匀,装入10ml试管中,每支装量1克,并塞上棉塞,用牛皮纸包好;砂土管经高压灭菌间歇5-6次,每次时间为1小时,消毒温度121~128℃,0.10~0.15Mpa,检测无菌则试验合格。
本发明灰黄霉素低产沫菌种的制备方法还包括留种保藏步骤:选取复筛后体积小、效价单位高的5~6个对应斜面孢子,用接种刀挑取一小块生长丰满、外观正常的斜面菌落置于三角瓶内,用吸管吸取灭菌水少量于三角瓶内,充分震荡,洗下孢子,制成悬浮液;将悬浮液接入砂土管内,每支砂土管接入0.2~0.3ml悬浮液,塞上棉塞,包上小纱布,绳子扎紧;放入真空干燥箱内,抽真空,取出砂土管移入冰箱,2~8℃保存。
本发明还公开了一种灰黄霉素低产沫菌种CGMCC NO.20257的应用,该灰黄霉素低产沫菌种CGMCC NO.20257用于发酵生产灰黄霉素。
实施例1:挑选已经培养筛选出的低产沫菌种,培养7~8天后观察现象。将培养成
熟的斜面和原孢子分别接入摇瓶中,于30±1℃温度条件下恒温振荡10-15天左右,取下摇
瓶测定体积并进行含量分析。
检测项目 | 原菌种 | 低产沫菌种 |
培养时间 | 15-16天 | 12-13天 |
外观 | 泡沫丰富且密集,泡沫约占整个培养瓶体积的60%左右 | 泡沫少,小且疏松,漂浮于表面,约占总体积10%左右 |
效价 | 28963mg/L | 29952mg/L |
实施例2:挑选已经培养筛选出的低产沫菌种,培养7~8天后观察现象。将培养成
熟的斜面和原孢子分别接入摇瓶中,于28±1℃温度条件下恒温振荡12-18天左右,取下摇
瓶测定体积并进行含量分析。
检测项目 | 原菌种 | 低产沫菌种 |
培养时间 | 17-18天 | 12-13天 |
外观 | 泡沫丰富且密集,泡沫约占整个培养瓶体积的65%左右 | 泡沫少,小且疏松,漂浮于表面,约占总体积15%左右 |
效价 | 27679mg/L | 28902mg/L |
由实施例1和实施例2可以看出,本发明最终制备出的灰黄霉素低产沫菌株,发酵培养过程中产沫量显著减少,发酵过程不再添加消泡剂,缩减操作步骤,降低能耗,进一步降低了生产成本,同时提高了菌株的单位效价并提高了收率。
最后应当说明的是,以上内容仅用以说明本发明的技术方案,而非对本发明保护范围的限制,本领域的普通技术人员对本发明的技术方案进行的简单修改或者等同替换,均不脱离本发明技术方案的实质和范围。
Claims (5)
1.一种灰黄霉素低产沫菌种,其特征在于:所述灰黄霉素低产沫菌种为展青霉(Penicillium patulum)GLINT 19-21-30-2#1,保藏编号为CGMCC NO.20257。
2.一种灰黄霉素低产沫菌种的制备方法,其特征在于:所述灰黄霉素低产沫菌种的制备方法具体包括如下步骤:
(1)用消好菌的接种针从砂土管内挑出少量展青霉菌4541的砂土孢子,反复、均匀地涂布在斜面培养基上,将接好种的斜面置于温度为28±1℃培养箱内培养7~8天;
(2)培养成熟后,挑选出外观为白色、孢子丰满、疏密均匀、边缘整齐、背面为红棕色的斜面,用消好菌的无菌玻璃珠将孢子打下,加入消好菌的纯化水20ml,再次充分振摇玻璃珠5分钟;
(3)取装有滤纸和药棉的玻璃漏斗过滤,过滤后取滤液,从中吸取2.0ml于消好菌的空白平碟中稀释作对照,另取滤液8~10ml于双碟中,用紫外线均匀照射15~20分钟,照毕,盖上双碟,用黑布罩住,并关闭净化台日光灯,借助缓冲间的灯光,以极快的速度稀释成浓度为10-5cfu/ml和10-6cfu/ml的稀释液,然后分别吸取浓度为10-5cfu/ml和10-6cfu/ml的稀释液于事先配制好的含有LiCl的分离培养基平板中,每皿0.1ml菌液,将同一浓度的平板用玻璃耙子耙匀分散后,移入28±1℃温度条件下恒温室培养7~8天;
(4)培养成熟后,分生孢子单菌落外观为大小均匀、丰富、厚实,孢子雪白且结合紧密,用接种针挑取分生孢子单菌落移入斜面试管,斜面培养基的配方与分离培养基配方相同,并注明剂量和编号,移至28±1℃温度条件下恒温室培养;
(5)培养7~8天后,斜面雪白一片、表面孢子结合紧密、背部色素为浅色,将培养成熟的菌落接入摇瓶中,并于30±1℃温度条件下恒温振荡12~13天,取下摇瓶测定体积并进行含量分析,选取体积小、效价单位高于初始菌种3%的15~20个斜面,转接斜面并编号;
(6)用已选取编号的斜面,通过挖块进行摇瓶复筛,培养和接种工艺同上一步骤;
(7)培养基不变,初筛后还需经过2-3次复筛培养,才能够得到灰黄霉素低产沫菌种CGMCC NO.20257。
3.根据权利要求1所述的一种灰黄霉素低产沫菌种的制备方法,其特征在于:所述灰黄霉素低产沫菌种的制备方法还包括砂土管的制备步骤:取黄沙和黄土适量,黄沙和黄土用盐酸溶液浸泡24小时后倒出,用纯化水冲洗至pH=7,烘干;将经过处理的黄沙和黄土以1:1的体积比混合均匀,装入10ml试管中,每支装量1克,并塞上棉塞,用牛皮纸包好;砂土管经高压灭菌间歇5-6次,每次时间为1小时,消毒温度121~128℃,0.10~0.15Mpa,检测无菌则试验合格。
4.根据权利要求1所述的一种灰黄霉素低产沫菌种的制备方法,其特征在于:所述灰黄霉素低产沫菌种的制备方法还包括留种保藏步骤:选取复筛后体积小、效价单位高的5~6个对应斜面孢子,用接种刀挑取一小块生长丰满、外观正常的斜面菌落置于三角瓶内,用吸管吸取灭菌水少量于三角瓶内,充分震荡,洗下孢子,制成悬浮液;将悬浮液接入砂土管内,每支砂土管接入0.2~0.3ml悬浮液,塞上棉塞,包上小纱布,绳子扎紧;放入真空干燥箱内,抽真空,取出砂土管移入冰箱,2~8℃保存。
5.一种灰黄霉素低产沫菌种CGMCC NO.20257的应用,其特征在于:所述灰黄霉素低产沫菌种CGMCC NO.20257用于发酵生产灰黄霉素。
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