CN108277169A - 一种快速筛选高产洛伐他汀红曲霉的方法 - Google Patents
一种快速筛选高产洛伐他汀红曲霉的方法 Download PDFInfo
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Abstract
本发明公开了一种快速筛选高产洛伐他汀红曲霉的方法,利用微孔板高通量培养技术(固态及液态)培养红曲霉,同时基于酶标仪以双波长法(△A=A238‑A247)快速检测发酵液中洛伐他汀含量。相比于常规平板培养和摇瓶发酵,本发明中所述的微孔板高通量培养技术试样量小、一次性可处理样品种类多,且节省空间,培养箱利用率高;对于高产洛伐他汀菌株的快速筛选,相比于液相色谱法,本发明实验周期大大缩短;相比于文献中所报道的比色法(单波长或双波长),本发明更能排除发酵液中其他物质的干扰。总之,整个过程可同时对上百株红曲霉进行快速培养和筛选,整个过程工作量小,操作快速便捷,易于实现。
Description
技术领域
本发明属于红曲霉筛选技术领域,具体涉及一种快速筛选高产洛伐他汀红曲霉的方法。
背景技术
红曲霉是从红曲中分离纯化出的黄酒酿造用菌,红曲霉会产很多有益的代谢产物,洛伐他汀就是其中一种,具有抑制胆固醇合成、降血脂等功效。筛选高产洛伐他汀的红曲霉的目的主要在于制备功能性红曲粉,使红曲色素中富含较高含量的洛伐他汀。
在实验室的研究中,红曲霉的筛选一般需要经过平板活化、摇瓶发酵。近期微孔板发酵技术的兴起,微孔板逐渐替代摇瓶发酵而作为一种高通量培养、筛选的技术,但前期红曲霉的扩培仍停留在平板活化的旧技术上。另一方面,经过高通量培养技术后,新型的快速检测技术尚待建立,多数文献关于产洛伐他汀菌的快速筛选方法都基于液相色谱检测技术,液相色谱虽然精确,但相对耗时,且随着检测时间的增长,排在后面的待测样品难免会发生变性;有学者采用比色法对洛伐他汀进行快速筛选,但单波长法(A238)的干扰性太强,难以说明此波长处的物质就是洛伐他汀,另外有学者发明了双波长法(△A=A246-A254)对洛伐他汀进行快速检测,但该方法仅仅排除了红曲色素的干扰而没有针对发酵液中其他物质进行干扰排除,而实际上,不含洛伐他汀的红曲发酵液中△A=A246-A254难以趋近于0,使得该方法的使用受到限制。
为克服上述缺点,本发明对红曲霉的培养过程(固态、液态)全部以微孔板进行高通量培养,同时重新构建新的双波长检测技术,以微孔板为载体,酶标仪为检测仪器进行快速检测。
发明内容
本发明的目的在于针对现有技术不足,提供一种快速筛选高产洛伐他汀红曲霉的方法。
为实现上述目的,本发明采用如下技术方案:
一种快速筛选高产洛伐他汀红曲霉的方法:在微孔板PDA培养基上对红曲霉进行活化,再将固体培养基转接至深孔板种子液中30℃、300r/min培养48h,再将深孔板种子液转接至深孔板发酵液中30℃、300r/min培养6d;取一定量发酵液于微孔板中加70wt%乙醇抽提洛伐他汀,对孔板进行离心过滤后移取上清液于96孔板中进行双波长检测;整个培养及检测过程全都以微孔板或深孔板为载体;整个操作过程具体包括以下步骤:
(1)红曲霉固态培养:取1.5mL PDA培养基于24孔微孔板中,将红曲霉在PDA固体培养基上进行单菌落点种并培养6~7d;
(2)孢子液的洗脱:在培养后的微孔板中每孔加入0.5~0.7mL生理盐水,于恒温振荡器中30℃、1200r/min 振荡5min,使孢子洗脱下来;
(3)种子液的培养:取上述孢子液接种于每孔装有5mL的液体种子培养基的24孔深孔板中,孢子液接种量为4~10%(v/v),在28~30℃、300r/min条件下培养36~48h;所述液体种子培养基的制备方法为:籼米粉35.00g,无水葡萄糖12.00g,黄豆粉12.00g,硝酸钠1.00g,磷酸二氢钾1.00,七水合硫酸镁0.50g,加去离子水定容至1000mL,自然pH;
(4)发酵液的培养:将步骤(3)培养后的种子液接入每孔装有5mL的液体发酵培养基的24孔深孔板中,种子液接种量为2~4%(v/v),在28~30℃、300r/min条件下培养6~7d,所述液体发酵培养基的制备方法为:味精22.33g,硫酸铵9.72g,籼米粉45g,无水葡萄糖12.50g,磷酸二氢钾9.00g,一水合硫酸锰0.21g,七水合硫酸镁2.1g,加去离子水定容至1000mL,自然pH;
(5)双波长法检测:在24孔深孔板中每孔加入0.2mL步骤(4)培养得到的发酵液和3.8mL的70wt%乙醇,在500r/min、60℃条件下旋转震荡2h,震荡后用孔板离心机4000r/min离心30min;取上清液于96孔板中,于酶标仪上进行双波长检测,检测波长为△A=A238-A247;以洛伐他汀标品为对照绘制双波长标曲,计算样品中洛伐他汀含量。
此外,步骤(3)中所述的液体种子培养基的制备方法还可以为:淀粉3.0g/L,硝酸钠2.5g/L,磷酸二氢钾2.5g/L,硫酸镁1.0g/L,黄豆粉1.0g/L,玉米浆干粉1.0g/L,pH 4.3,121℃灭菌20min。
步骤(4)中所述的液体发酵培养基的制备方法还可以为:葡萄糖40.0g/L,蛋白胨5.0g/L,硫酸镁1.0g/L,硝酸钠3.0g/L,磷酸二氢钾1.5g/L,pH 4~4.5,121℃灭菌20min。
本发明的有益效果在于:
(1)以微孔板替代平板活化和摇瓶培养,不仅节省培养基原料,而且大大提高了培养箱及摇床的使用效率,可一次性培养数百株红曲霉;
(2)基于微孔板和酶标仪的双波长法测定发酵液中的洛伐他汀含量,克服了液相色谱法的耗时长的缺点,同时对文献中的双波长法进行改进,使得检测结果更贴近实际,避免了发酵液中洛伐他汀以外的物质的干扰,该检测方法可以做到定性半定量分析。
附图说明
图1为微孔板PDA固态培养;
图2为深孔板液态发酵培养;
图3为微孔板离心;
图4为微孔板检测;
图5为含洛伐他汀溶液与不含洛伐他汀的红曲发酵液全波长扫描图;
图6为10μg/mL的洛伐他汀液相色谱图;
图7为无洛伐他汀红曲发酵液的液相色谱图;
图8为双波长下洛伐他汀标准曲线;
图9为双波长法与液相色谱法检测结果对比图。
具体实施方式
以下结合具体实施例对本发明做进一步说明,但本发明不仅仅限于这些实施例。
实施例1
一种快速筛选高产洛伐他汀红曲霉的方法,以微孔板高通量培养技术培养红曲霉,同时基于酶标仪以双波长法快速检测发酵液中洛伐他汀含量,具体包括以下步骤:
(1)红曲霉固态培养:取1.5mL PDA培养基于24孔微孔板中,将红曲霉在PDA固体培养基上进行单菌落点种并培养7d;
(2)孢子液的洗脱:在培养后的微孔板中每孔加入0.7mL生理盐水,于恒温振荡器中30℃、1200r/min 振荡5min,使孢子洗脱下来;
(3)种子液的培养:取上述孢子液接种于每孔装有5mL的液体种子培养基的24孔深孔板中,孢子液接种量为4%(v/v),在30℃、300r/min条件下培养48h;所述,液体种子培养基的制备方法为:籼米粉35.00g,无水葡萄糖12.00g,黄豆粉12.00g,硝酸钠1.00g,磷酸二氢钾1.00,七水合硫酸镁0.50g,加去离子水定容至1000mL,自然pH;
(4)发酵液的培养:将步骤(3)培养后的种子液接入每孔装有5mL的液体发酵培养基的24孔深孔板中,种子液接种量为4%(v/v),在30℃、300r/min条件下培养6d;所述液体发酵培养基的制备方法为:味精22.33g,硫酸铵9.72g,籼米粉45g,无水葡萄糖12.50g,磷酸二氢钾9.00g,一水合硫酸锰0.21g,七水合硫酸镁2.1g,加去离子水定容至1000mL,自然pH;
(5)双波长法检测:在24孔深孔板中每孔加入0.2mL步骤(4)培养得到的发酵液和3.8mL的70wt%乙醇,在500r/min、60℃条件下旋转震荡2h,震荡后用孔板离心机4000r/min离心30min;取上清液于96孔板中,于酶标仪上进行双波长检测,检测波长为△A=A238-A247。以洛伐他汀标品为对照绘制双波长标曲,计算样品中洛伐他汀含量。
图5为上述样品中测得的含洛伐他汀溶液与不含洛伐他汀的红曲发酵液全波长扫描图,从图中可以看出,洛伐他汀在228nm、238nm、247nm处有最大吸收峰,尽管稀释后的红曲发酵液在紫外波段吸光值很高,但在双波长处△A=A238-A247=0.0073≈0,而洛伐他汀的△A=A238-A247=0.3482。显然此双波长法适用于检测红曲发酵液中的洛伐他汀。若采用文献中所述的双波长法,不含洛伐他汀的红曲发酵液中△A=A246-A254=0.1034,则容易将不产洛伐他汀的菌株误判为产洛伐他汀菌株,不利于生产应用。
针对实验中的红曲发酵液是否真的不含洛伐他汀,有液相色谱检测结果为证。
液相色谱法:色谱柱C18(4.6*250mm,5mm);流动相:甲醇:0.02%磷酸(V:V)=75:25;流速:1.0mL/min,柱温30℃,波长:237nm,进样量:20μL,检测时间:40min;测试结果如图6、7所示,所用红曲发酵液的液相色谱图中保留时间15.4min附近无出峰,说明该样品中无洛伐他汀。此外,如图8所示,双波长下洛伐他汀标曲线形良好,说明该方法具有可行性。
对上述无洛伐他汀的红曲发酵液中进行加标检测,双波长法与液相色谱法对比情况如图9所示,回归方程的斜率为0.9574<1,说明酶标法的检测值略微小于液相色谱的检测值,但酶标法检测与液相色谱法检测的结果线性良好,说明两者之间具有很好的线性关系。基于快速筛选的目的上,酶标法具有很好的实用性和一定程度上的准确性。
因此,本发明能够准确、灵敏的测定红曲发酵液中洛伐他汀的含量,并且不容易造成误判,从而能够快速筛选高产洛伐他汀的红曲霉。
实施例2
改变红曲霉的种子液配方,其余实施方式不变。
例如红曲霉种子液配方改为:淀粉3.0g/L,硝酸钠2.5g/L,磷酸二氢钾2.5g/L,硫酸镁1.0g/L,黄豆粉1.0g/L,玉米浆干粉1.0g/L,pH 4.3,121℃灭菌20min。
实施例3
改变红曲霉的发酵液配方,其余实施方式不变。
例如红曲霉发酵液配方改为:葡萄糖40.0g/L,蛋白胨5.0g/L,硫酸镁1.0g/L,硝酸钠3.0g/L,磷酸二氢钾1.5g/L,pH 4~4.5,121℃灭菌20min。
实施例4
改变接种量,其余实施方式不变。
例如:红曲霉孢子液的接种量改为10%,种子液的接种量改为2%。
实施例5
改变孢子洗脱方式,其余实施方式不变。
例如:在含有PDA培养基的微孔板中加入0.5mL生理盐水,用接种铲将孢子刮下,取孢子悬液于种子培养液中。
实施例6
改变培养时间,其余实施方式不变。
例如:PDA培养周期为6天,种子液培养时间为36h,发酵液培养时间为7d。
实施例7
改变培养温度,其余实施方式不变。
例如:将培养温度30℃改为28℃。
实施例8
改变检测前样品前处理方式,其余实施方式不变。
例如:将发酵结束后的发酵液直接4000r/min离心30min,取上清液于96孔板中于酶标仪上进行双波长检测,检测波长为△A=A238-A247。以洛伐他汀标品为对照绘制双波长标曲,计算样品中洛伐他汀含量。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (5)
1.一种快速筛选高产洛伐他汀红曲霉的方法,其特征在于:以微孔板高通量培养技术培养红曲霉,同时基于酶标仪以双波长法快速检测发酵液中洛伐他汀含量,具体包括以下步骤:
(1)红曲霉固态培养:取1.5mL PDA培养基于24孔微孔板中,将红曲霉在PDA固体培养基上进行单菌落点种并培养6~7d;
(2)孢子液的洗脱:在培养后的微孔板中每孔加入0.5~0.7mL生理盐水,于恒温振荡器中30℃、1200r/min 振荡5min,使孢子洗脱下来;
(3)种子液的培养:取上述孢子液接种于每孔装有5mL的液体种子培养基的24孔深孔板中,孢子液接种量为4~10%(v/v),在28~30℃、300r/min条件下培养36~48h;
(4)发酵液的培养:将步骤(3)培养后的种子液接入每孔装有5mL的液体发酵培养基的24孔深孔板中,种子液接种量为2~4%(v/v),在28~30℃、300r/min条件下培养6~7d;
(5)双波长法检测:在24孔深孔板中每孔加入0.2mL步骤(4)培养得到的发酵液和3.8mL的70wt%乙醇,在500r/min、60℃条件下旋转震荡2h,震荡后用孔板离心机4000r/min离心30min;取上清液于96孔板中,于酶标仪上进行双波长检测,检测波长为△A=A238-A247;以洛伐他汀标品为对照绘制双波长标曲,计算样品中洛伐他汀含量。
2.根据权利要求1所述的一种快速筛选高产洛伐他汀红曲霉的方法,其特征在于:步骤(3)中所述的液体种子培养基的制备方法为:籼米粉35.00g,无水葡萄糖12.00g,黄豆粉12.00g,硝酸钠1.00g,磷酸二氢钾1.00,七水合硫酸镁0.50g,加去离子水定容至1000mL,自然pH。
3. 根据权利要求1所述的一种快速筛选高产洛伐他汀红曲霉的方法,其特征在于:步骤(3)中所述的液体种子培养基的制备方法为:淀粉3.0g/L,硝酸钠2.5g/L,磷酸二氢钾2.5g/L,硫酸镁1.0g/L,黄豆粉1.0g/L,玉米浆干粉1.0g/L,pH 4.3,121℃灭菌20min。
4.根据权利要求1所述的一种快速筛选高产洛伐他汀红曲霉的方法,其特征在于:步骤(4)中所述的液体发酵培养基的制备方法为:味精22.33g,硫酸铵9.72g,籼米粉45g,无水葡萄糖12.50g,磷酸二氢钾9.00g,一水合硫酸锰0.21g,七水合硫酸镁2.1g,加去离子水定容至1000mL,自然pH。
5. 根据权利要求1所述的一种快速筛选高产洛伐他汀红曲霉的方法,其特征在于:步骤(4)中所述的液体发酵培养基的制备方法为:葡萄糖40.0g/L,蛋白胨5.0g/L,硫酸镁1.0g/L,硝酸钠3.0g/L,磷酸二氢钾1.5g/L,pH 4~4.5,121℃灭菌20min。
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