CN109112081A - 一种牛蒡根固体链霉菌产孢培养基及培养方法 - Google Patents
一种牛蒡根固体链霉菌产孢培养基及培养方法 Download PDFInfo
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Abstract
本发明公开了一种牛蒡根固体链霉菌产孢培养基及链霉菌培养的方法,培养基由干牛蒡根20~30g/L、甘露醇15~25g/L、琼脂粉15~20g/L和水组成。将牛蒡根烘干粉碎后,加入水煮沸后过滤,滤液与甘露醇、琼脂粉混合,用NaOH调节pH值至7.0~7.5,定容,灭菌后倒入平皿,凝固后利用接种环进行划线培养链霉菌。本发明原料少,配制简单,简化了产孢培养基的配制方法,成本低廉,中牛蒡根中氨基酸量约占干重的28%,菊糖含量约占干重的34%,且牛蒡中多种微量元素,其与放线菌培养基高氏一号培养基比较,链霉菌能够快速增殖,产孢质量好,通过生物抑菌活性测定表明,在本发明培养基中生长的链霉菌抑制金黄色葡萄球菌和红酵母的生物活性明显优于高氏一号培养基。
Description
技术领域
本发明属于微生物固体培养技术领域,具体的说,涉及一种牛蒡根固体链霉菌产孢培养基及其应用。
背景技术
链霉菌是抗生素来源的重要生物资源,具有复杂的生长繁殖过程,在其代谢过程中可以产生多种抗菌活性天然产物。在实验室研究及抗生素生产过程中需要对链霉菌进行培养,获得丰富的孢子,以及筛选可以产生生物活性物质的菌株,满足生产及科研的需求。
牛蒡(Arctium lappa)属菊科牛蒡属,二年生草本植物,又名东洋参、大力子等,是中国传统药食同源的植物,而且牛蒡的收获期长,耐贮藏。牛蒡在我国种植历史较久,分布广泛,产量较高,在欧洲、北美也有几百年的牛蒡药用历史。目前,牛蒡的经济价值主要体现在牛蒡根作为一种新特蔬菜出口日本、韩国以及欧洲许多国家。
牛蒡中含有丰富的营养成分,牛蒡根呈纺锤形,肉质而直立。目前,从牛蒡根中发现的活性成分主要有氨基酸类、微量元素、糖类、多酚类(咖啡酸、绿原酸等)、炔类、挥发油、黄酮类、糖苷类、生物碱、牛蒡苦素、丁二酸、齐墩果酸、熊果酸、胡萝卜苷等。其应用前景广阔,可对其进行进一步开发,生产出具有高附加值的产品。将牛蒡根用于微生物培养,可以有效的拓宽牛蒡根的用途,提高牛蒡根的利用价值。
发明内容
本发明涉及一种牛蒡根固体链霉菌产孢培养基及链霉菌培养的方法,提高牛蒡的利用价值。
一种牛蒡根固体链霉菌培养基,其特征在于:包括牛蒡根20~30g/L、甘露醇15~25g/L、琼脂粉15~20g/L和水。所述牛蒡根固体链霉菌培养基的方法,其特征在于,包括以下步骤:将牛蒡根烘干,称取干牛蒡根,粉碎后,加入水煮沸,纱布过滤,滤液与甘露醇、琼脂粉混合,用NaOH调节pH值至7.0~7.5,定容,灭菌后倒入平皿,凝固后使用。
应用所述一种牛蒡根固体链霉菌培养基的方法,其特征在于,利用接种环沾取链霉菌进行划线,将链霉菌产孢平皿置于恒温培养箱中培养,得到链霉菌孢子。
更具体的:一种牛蒡根固体产孢培养基培养链霉菌的方法,包括以下步骤:产孢培养基的配制:将牛蒡根洗净,置于烘箱中80℃烘干,称取20~30g干牛蒡根,用粉碎机粉碎,加入15~25g甘露醇,加入600~800ml水充分混匀后,利用NaOH调节pH至7.0~7.5,加入15~20g琼脂粉,最后加水定容至1L体积,灭菌后分装至平皿中,凝固后使用。
链霉菌孢子的培养:取链霉菌菌种,用无菌接种环在牛蒡根固体培养基中划线培养,得到丰富的链霉菌孢子。
进一步地,所述培养基灭菌具体为:103.4KPa,121℃高压蒸汽灭菌30min。
进一步地,所述牛蒡根固体链霉菌产孢培养基培养链霉菌的方法,其特征在于,所述链霉菌培养温度为25~30℃。
进一步地,所述的牛蒡根固体链霉菌产孢培养基培养链霉菌的方法,其特征在于,所述链霉菌培养时间为5~10天。
本发明的技术优势为:使用牛蒡根代替了产孢培养基中的氮源和微量元素,减少培养基中有机氮源的用量及种类,配制方法快速简单。产孢培养基使用牛蒡根配制,营养丰富,有利于链霉菌产孢,提高孢子质量。同时,将该产孢培养基培养的链霉菌进行抑菌活性测试,所培养链霉菌抑制金黄色葡萄球菌和红酵母的活性明显优于高氏一号培养基培养链霉菌的活性,有利于产活性物质链霉菌的筛选。利用干牛蒡根作为原料,保证了原料来源充足稳定。
附图说明
图1Streptomyces cala在不同干牛蒡根培固体养基中生长状况
图2不同链霉菌在高氏一号培养基和牛蒡根固体产孢培养基中生长状况
图3生物活性分析结果,A中1为在高氏一号培养基生长的Streptomycesviolaceus S21,2为在牛蒡根固体产孢培养基中生长的Streptomyces violaceus S21,指示菌为金黄色葡萄球菌;B中3为在高氏一号培养基生长的Streptomyces FR008,4为在牛蒡根固体产孢培养基中生长的Streptomyces FR008,指示菌红酵母
具体实施方式
下面用实例予以说明本发明,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
一种牛蒡根固体培养基培养链霉菌Streptomyces cala的方法:
配制1L产孢培养基:分别称取10g、20g、30g干牛蒡根,粉碎后,加入600ml水煮沸三次,四层纱布过滤,加入20g甘露醇、20g琼脂粉混合,用NaOH调节pH值至7.0,定容至1L,分别编号A、B、C,所述培养基灭菌具体为:103.4KPa,121℃高压蒸汽灭菌30min。
在超净工作台内将A、B、C三种灭菌后的产孢培养基分别倒入平皿,待培养基凝固后,在超净工作台内操作,利用接种环将链霉菌Streptomyces cala进行划线培养。
将划线后的链霉菌平皿倒置于恒温培养中培养,孢子培养过程中,培养温度为28℃,培养时间为7天。结果表明:Streptomyces cala在B(干牛蒡根20g)、C(干牛蒡根30g)固体链霉菌培养基中产孢良好,如图1所示,因此优选的将干牛蒡根含量定为20~30g。
实施例1
一种牛蒡根固体培养基培养链霉菌产孢的方法:
配制1L产孢培养基:称取25g干牛蒡根,粉碎后,加入600ml水煮沸三次,四层纱布过滤,加入20g甘露醇、20g琼脂粉混合,用NaOH调节pH值至7.0,定容至1L,103.4KPa,121℃高压蒸汽灭菌30min。
在超净工作台内将灭菌后的产孢培养基倒入平皿,待培养基凝固后,在超净工作台内操作,利用接种环进行划线培养链霉菌。
将划线后的链霉菌平皿倒置于恒温培养中培养,孢子培养过程中,培养温度为28℃,培养时间为10天。结果表明:链霉菌:Streptomyces FR008、Streptomyces M145、Streptomyces cala、Streptomyces MHY21、Streptomyces violaceus S21均能在牛蒡根固体链霉菌培养基生长产孢,效果明显优于高氏一号培养基,如图2所示。
实施例2
一种牛蒡根固体培养基筛选产活性物质链霉菌的方法:
配制1L产孢培养基:称取25g干牛蒡根,粉碎后,加入600ml水煮沸三次,四层纱布过滤,加入20g甘露醇、20g琼脂粉混合,用NaOH调节pH值至7.0,定容至1L,103.4KPa,121℃高压蒸汽灭菌30min。
在超净工作台内将灭菌后的产孢培养基倒入平皿,待培养基凝固后,在超净工作台内操作,利用接种环将链霉菌孢子均匀涂布于牛蒡根固体培养基中。
将涂布链霉菌的平皿倒置于恒温培养中培养,培养温度为28℃,培养时间为7天。
利用打孔器在平皿里打取菌块,将琼脂菌块置于接有指示菌(金黄色葡萄球菌或红酵母)的平皿中,将指示菌平皿置于恒温培养箱中培养(金黄色葡萄球菌37℃培养16h,红酵母28℃培养48h),观察抑菌情况。
生物活性测定结果表明:在牛蒡根固体培养基上培养的链霉菌Streptomycesviolaceus S21对金黄色葡萄球菌具有明显的抑菌活性,而在高氏一号培养基中生长的Streptomyces violaceus S21活性微弱(图3A);在牛蒡根固体培养基上培养的链霉菌Streptomyces FR008对红酵母的抑菌圈明显比在高氏一号培养基中的抑菌圈大(图3B)所示。
Claims (4)
1.一种牛蒡根固体链霉菌培养基,其特征在于:包括牛蒡根20~30g/L、甘露醇15~25g/L、琼脂粉15~20g/L和水。
2.制备如权利要求1所述一种牛蒡根固体链霉菌培养基的方法,其特征在于,包括以下步骤:
将牛蒡根烘干,称取干牛蒡根,粉碎后,加入水煮沸,纱布过滤,滤液与甘露醇、琼脂粉混合,用NaOH调节pH值至7.0~7.5,定容,灭菌后倒入平皿,凝固后使用。
3.如权利要求2所述的方法,其特征在于,所述灭菌具体为:103.4KPa,121℃高压蒸汽灭菌30min。
4.应用如权利要求1所述一种牛蒡根固体链霉菌培养基的方法,其特征在于,利用接种环沾取链霉菌进行划线,将链霉菌产孢平皿置于恒温培养箱中培养,得到链霉菌孢子,所述链霉菌培养温度为25~30℃;链霉菌培养时间为5~10天。
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