CN106854622B - 一种辐照灭菌后pH稳定的培养基平板 - Google Patents

一种辐照灭菌后pH稳定的培养基平板 Download PDF

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CN106854622B
CN106854622B CN201611226318.7A CN201611226318A CN106854622B CN 106854622 B CN106854622 B CN 106854622B CN 201611226318 A CN201611226318 A CN 201611226318A CN 106854622 B CN106854622 B CN 106854622B
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杨宁
李斌
卢勉飞
蔡芷荷
李泽康
吴清平
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Guangdong Huankai Microbial Sci and Tech Co Ltd
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Abstract

本发明公开了一种辐照灭菌后pH稳定的培养基平板，培养基由胨、酵母浸出粉、葡萄糖、琼脂和抗氧化剂和水组成。本发明的YPD平板5KGy剂量辐照的最终灭菌后，pH值较为稳定，20～25℃的阴凉库贮存4个月后pH未见明显变化，并且生物学特性未受明显影响，培养基性能依旧良好，大大延长了产品的货架期。

Description

一种辐照灭菌后pH稳定的培养基平板
技术领域
本发明涉及一种平板，特别涉及一种辐照灭菌后pH稳定的培养基平板。
背景技术
中国药典2015年版通则9203药品微生物实验室质量管理指导原则规定：“用于环境监测的培养基须特别防护，最好要双层包装和终端灭菌”，目前市面上用于环境监测的成品培养基普遍采用三层无菌包装和终端辐照灭菌的方式。而用于终端产品检测的成品培养基为保障无菌，也可采用终端辐照灭菌的方式。
但是，辐照会导致培养基理化性质的改变(如培养基颜色的改变、pH和凝胶强度的降低)甚至是营养成分的破坏。尤其是含糖量较高的培养基(比如YPD平板，酵母浸出粉胨葡萄糖琼脂培养基平板)pH下降幅度更为明显。这是因为辐照对于糖分子有水解和氧化降解的作用,引起辐解产物的形成，导致pH下降。辐照多糖会引发解聚。辐照单糖水溶液会产生H2、CO、CO2、甲醛、丙醛、乙二醛、醛糖糖酸、糖醛酸、糖聚合体、脱氧化合物等一系列对微生物生长有影响的物质。
实践证明，经辐照的成品培养基在有效期内pH值可能已低于标准要求下限，这大大缩短了产品的货架期，影响产品的使用寿命。
如何在不影响微生物生长的情况下，使平板的pH在进行辐照灭菌后依然保持稳定，是一件非常具有挑战性的工作，同时也一件十分有价值的工作。
发明内容
本发明的目的在于提供一种辐照灭菌后pH稳定的培养基平板。
本发明所采取的技术方案是：
一种辐照灭菌后pH稳定的培养基平板，培养基由胨、酵母浸出粉、葡萄糖、琼脂和抗氧化剂和水组成。
作为上述培养基平板的进一步改进，每1000ml培养基中含有胨5～20g，酵母浸出粉3～10g，葡萄糖10～40g，琼脂9～20g和0.1～5g的抗氧化剂。
作为上述培养基平板的进一步改进，每1000ml培养基中含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、0.3～3g的抗氧化剂。
作为上述培养基平板的进一步改进，抗氧化剂选自硫酸亚铁、柠檬酸钠、硫代硫酸钠和亚硫酸氢钠中的至少一种，更佳的，抗氧化剂为亚硫酸氢钠。
作为上述培养基平板的进一步改进，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、亚硫酸氢钠1.1～1.3g。
作为上述培养基平板的进一步改进，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、亚硫酸氢钠1.2g。
本发明的有益效果是：
本发明的YPD平板5KGy剂量辐照的最终灭菌后，pH值较为稳定，20～25℃的阴凉库贮存4个月后pH未见明显变化，并且生物学特性未受明显影响，培养基性能依旧良好，大大延长了产品的货架期。
具体实施方式
一种辐照灭菌后pH稳定的培养基平板，培养基由胨、酵母浸出粉、葡萄糖、琼脂和抗氧化剂和水组成。
作为上述培养基平板的进一步改进，每1000ml培养基中含有胨5～20g，酵母浸出粉3～10g，葡萄糖10～40g，琼脂9～20g和0.1～5g的抗氧化剂。
作为上述培养基平板的进一步改进，每1000ml培养基中含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、0.3～3g的抗氧化剂。
作为上述培养基平板的进一步改进，抗氧化剂选自硫酸亚铁、柠檬酸钠、硫代硫酸钠和亚硫酸氢钠中的至少一种，更佳的，抗氧化剂为亚硫酸氢钠。
作为上述培养基平板的进一步改进，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、亚硫酸氢钠1.1～1.3g。
作为上述培养基平板的进一步改进，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、亚硫酸氢钠1.2g。
下面结合实施例，进一步说明本发明的技术方案。
实施例1
一种培养基平板，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、亚硫酸氢钠1.2g。
实施例2
一种培养基平板，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、硫代硫酸钠1.2g。
实施例3
一种培养基平板，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、硫酸亚铁1.2g。
实施例4
一种培养基平板，每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、柠檬酸钠1.2g。
实施例5
一种培养基平板，每1000ml培养基含有胨5.0g、酵母浸出粉10.0g、葡萄糖40.0g、琼脂9.0g、亚硫酸氢钠5.0g。
实施例6
一种培养基平板，每1000ml培养基含有胨15.0g、酵母浸出粉4.0g、葡萄糖15.0g、琼脂16.0g、亚硫酸氢钠1.1g。
实施例7
一种培养基平板，每1000ml培养基含有胨12.0g、酵母浸出粉7.0g、葡萄糖30.0g、琼脂12.0g、亚硫酸氢钠1.3g。
实施例8
一种培养基平板，每1000ml培养基含有胨20.0g、酵母浸出粉3.0g、葡萄糖10.0g、琼脂20.0g、亚硫酸氢钠0.1g。
平板性能测试：
1材料
1.1菌株
酿酒酵母(Saccharomyces cerevisiae)ATCC9763，白色念珠菌(Candidaalbicans)CMCC(F)98001由本实验室保存。当然，也可以使用其他可以公开获得的同种菌株进行测试。
1.2培养基及试剂
实施例1所述YPD平板(简称YPD平板1)、实施例2所述YPD平板(简称YPD平板2)、实施例3所述YPD平板(简称YPD平板3)、实施例4所述YPD平板(简称YPD平板4)、原配方YPD平板(不含抗氧化物)和沙氏葡萄糖琼脂培养基平板(简称SDA平板)由广东环凯微生物科技有限公司提供。酵母浸出粉胨葡萄糖琼脂培养基标准品由中国食品药品检定研究院提供。
2方法
2.1培养基pH稳定性能比较
2.1.1YPD平板制备：
按酵母浸出粉胨葡萄糖琼脂培养基标准配方称取各原料(原配方平板)，并按比例加入抗氧化物亚硫酸氢钠(YPD平板1)，或硫代硫酸钠(YPD平板2)，或硫酸亚铁(YPD平板3)，或柠檬酸钠(YPD平板4)，调整pH值，煮沸溶解，121℃灭菌20min，冷却至50℃倾注平板，备用。
2.1.2将制备好的上述各种YPD平板进行三层包装，送交辐照中心进行5KGy剂量的电子束辐照，以不进行辐照的平板作为对照。
辐照后置于20～25℃阴凉库储存，分别于辐照后第1天、第7天、第15天、第30天、第60天、第90天和第120天测定pH值。
2.2培养基生物学性能评价
生长率试验：采用定量方法进行。将白色念珠菌、酿酒酵母2株菌的新鲜斜面培养物制成约103cfu/ml的菌悬液。分别取上述各菌液0.1ml用全自动螺旋接种仪分别接种于经5KGy辐照的YPD平板1及未经辐照作为对照的原配方平板和酵母浸出粉胨葡萄糖琼脂培养基标准品上，白色念珠菌、酿酒酵母的菌液同时接种参比培养基SDA平板，23-28℃培养3～5d。每种平板进行3个重复。观察各菌株在培养基上的生长情况，并对其进行菌落计数。按下列公式计算生长率PR：
PR＝NS/N0，NS：待测培养基平板上得到的平均菌落数；N0：参比培养基平板上获得的平均菌落数。
3结果
3.1培养基pH值稳定性能比较
各配方培养基pH稳定性能比较见表1：
表1各配方培养基pH稳定性能比较
从表中结果可以看出，原配方YPD平板、YPD平板2、YPD平板3和YPD平板4辐照后pH值下降明显(下降值分别为0.49、0.22、0.20和0.18)，原配方YPD平板pH值下降更为明显，而YPD平板1辐照后未引起pH值下降，反而有略微上升；原配方YPD平板、YPD2平板、YPD平板3和YPD平板4辐照后4个月内pH值下降值分别为0.56、0.50、0.52和0.66，与辐照前相比，总的pH值下降值分别为1.05、0.72、0.72和0.84，而YPD平板1辐照后4个月内pH下降值为0.19，与辐照前相比，总的pH下降值为0.17。实施例1所述YPD平板pH值测定结果符合标准6.0±0.2的要求，pH变化值也在±0.2的范围，而其他四种平板已低于标准6.0±0.2的下限要求，pH变化值也在±0.2的范围外。总的来说，实施例1所述YPD平板的pH稳定性能优于其他四种YPD平板。
类似的，实施例5～8的培养基制备得到的平板在进行辐照灭菌后，pH也基本保持稳定。
3.2培养基生物学性能评价
结果见表2：
表2培养基生物学性能评价
从结果可以看出实施例1所述培养基经5KGy辐照后与未经辐照的原配方和中检所标准品相比，各目标菌的生长率无明显差异，目标菌白色念珠菌CMCC(F)98001和酿酒酵母ATCC9763均能达到0.7的生长率，且目标菌的菌落大小和形态与对照一致。表明实施例1所述培养基平板生物学性能良好，符合要求。
综上所述，实施例1所述的酵母浸出粉胨葡萄糖琼脂培养基平板经5KGy剂量辐照的最终灭菌后，pH值较为稳定，阴凉库贮存4个月后pH未见明显变化。并且生物学特性未受明显影响，培养基性能依旧良好。
虽然本发明对具体实施案例进行了描述，但是本发明并不局限于此。本行业技术人员应该意识到，在本发明所述原理下对本发明做的多种修饰和改变，均应认为是本发明的保护范围。

Claims (4)

1.一种辐照灭菌后pH稳定的培养基平板，培养基由胨、酵母浸出粉、葡萄糖、琼脂和抗氧化剂和水组成，其特征在于：每1000 ml培养基中含有胨5～20g，酵母浸出粉3～10 g，葡萄糖10～40 g，琼脂9～20g和0.1～5g的亚硫酸氢钠。
2.根据权利要求1所述的培养基平板，其特征在于：每1000 ml培养基中含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、0.3～3g的亚硫酸氢钠。
3.根据权利要求1所述的培养基平板，其特征在于：每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、亚硫酸氢钠1.1～1.3g。
4.根据权利要求1所述的培养基平板，其特征在于：每1000ml培养基含有胨10.0g、酵母浸出粉5.0g、葡萄糖20.0g、琼脂14.0g、亚硫酸氢钠1.2 g。
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""γ射线辐照培养基的灭菌效果及对微生物生长影响的研究"";钟士清 等;《广东农业科学》;19950531(第4期);29-31页

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