CN115094115B - 一种用于非无菌药品中间体微生物计数的快速检测方法 - Google Patents
一种用于非无菌药品中间体微生物计数的快速检测方法 Download PDFInfo
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Abstract
本发明涉及非无菌药品中间体微生物检测技术领域。本发明包括以下步骤:制备供试液;药典组:每个稀释级分别取1mL供试液加入胰酪大豆胨琼脂培养基中,平行制备2皿,置20~35℃培养3至7天;快检组:每个稀释级分别取1mL供试液加至需氧菌总数测试片,平行制备2片,置20~35℃培养2天;对比药典组合快检组得出结论。本发明最大程度减少系统适用性的改变,将微生物计数时长由7天缩短到2天。
Description
技术领域
本发明属于非无菌药品中间体微生物检测技术领域,具体是一种用于非无菌药品中间体微生物计数的快速检测方法。
背景技术
《中国药典》2020年版四部通则1105非无菌产品微生物限度检查:微生物计数法中对非无菌产品的微生物计数法的定义、操作及要求进行了规定。微生物计数法系用于能在有氧条件下生长的嗜温细菌和真菌的计数。计数方法包括平皿法、薄膜过滤法和最可能数法(Most-Probable-Number Method,筒称MPN法)。供试品检查时,应根据供试品理化特性和微生物限度标准等因素选择计数方法,检测的样品量应能保证所获得的试验结果能够判断供试品是否符合规定。
微生物计数法在《中国药典》2020年版四部通则或各论项下有详细编写。该方法的不足:1、培养周期较长,其中需氧菌总数需要培养3至5天,霉菌和酵母菌总数需要培养5至7天,难以应对药品突发事件对时间的紧迫要求,同时严重限制了企业内部生产中间体自检效率,延长了生产周期;2、在菌落计数时,受供试液中杂质、样品中不溶物的影响,会导致计数困难和准确性降低。为解决上述不足,在不改变企业原有非无菌药品终产品微生物计数的方法前提下,我们发明了一种用于非无菌药品中间体微生物计数快速检测的新方法。
发明内容
本发明的目的是针对以上问题,提供了一种用于非无菌药品中间体微生物计数的快速检测方法,本发明具有以下特点:具有快速、方便、灵敏度高、特异性强、操作简便、便于运输、成本低等优点,有良好的研究价值和应用前景。最大程度减少系统适用性的改变,将微生物计数时长由7天缩短到2 天。
为实现上述目的,本发明提供如下技术方案:一种用于非无菌药品中间体微生物计数的快速检测方法,包括以下步骤:
(1)依据2020版中国药典四部1106、1107制备供试液;
(2)药典组:每个稀释级分别取1mL供试液加入胰酪大豆胨琼脂培养基中,平行制备2皿,置20~35℃培养3至7天;
(3)快检组:揭开测试片的上层膜,取适供试液1ml加至测试片涂有计数显色培养基的镂空区域,使供试液均匀分布于整个培养区,将上层膜缓缓落下,避免挤压,静置30s~1min后放入恒温培养箱于20~35℃培养2天,按照2020版中国药典四部进行计数,每个稀释度平行制备两片,同时取1ml 生理盐水作空白对照;
(4)判定:药典组和快检组两组试验平行进行,培养结束后,观察菌落生长情况并计数,若测试片计数结果符合规定,则判定非无菌药品中间体菌总数合格;若测试片计数结果不符合规定,或平皿法计数结果不符合规定,则判定非无菌药品中间体菌总数不合格。
优选的,所述测试片包括用于测定需氧菌总数的需氧菌总数计数测试片和用于测定霉菌和酵母菌总数的霉菌和酵母菌总数计数测试片。
优选的,所述需氧菌总数的需氧菌总数计数测试片由如下步骤制备而成:
将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP底层裁剪成相同尺寸;将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP 底层的其中一边均留出3~5mm宽度粘合;将需氧菌冷水可溶凝胶粉末均匀地涂在PET复合膜的粘性面上;将需氧菌总数显色培养基与需氧菌冷水可溶性凝胶粉末按质量比8:(1~2)配制,再将得到的混合粉末均匀涂抹在PVC中层板的镂空区域内。
优选的,所述需氧菌总数显色培养基的制备:称取胰蛋白胨18~25g,大豆木瓜蛋白酶水解物5~12g,牛肉浸粉3~8g,氯化钠3~7g,葡萄糖2~6g,红四氮唑0.2~0.5g,单甘酯0.2~0.5g,混匀后得到需氧菌总数计数显色培养基。
优选的,所述需氧菌冷水可溶性凝胶粉末的制备:将经研磨后过100~150 目标准筛的黄原胶和瓜尔胶混合,得到需氧菌冷水可溶凝胶粉末,其中黄原胶和瓜尔胶的质量比为7:2~3。
优选的,所述霉菌和酵母菌总数计数测试片由如下步骤制备而成:将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP底层裁剪成相同尺寸;将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP底层的其中一边均留出3~5mm宽度粘合;将霉菌和酵母菌冷水可溶凝胶粉末均匀地涂在PET复合膜的粘性面上;将裁圆的灭菌无纺布浸泡于霉菌和酵母菌总数计数显色培养基中10~20分钟后取出,在无菌条件下烘干,贴于PVC中层板的镂空区域内。
优选的,所述霉菌和酵母菌总数计数显色培养基的制备:称取胰蛋白胨 15~25g,大豆木瓜蛋白酶水解物5~10g,葡萄糖15~20g,氯化钠2~8g,硫酸镁0.1~0.3g,硫酸铜0.1~0.3g,加入1000ml纯化水中,经121℃,15min 高压灭菌后冷却至室温,再加入滤菌后的氯霉素0.1~0.5g、5-溴-4-氯-3- 吲哚基-N-乙酰基-β-D-氨基半乳糖苷0.2~0.4g,制得霉菌和酵母菌总数计数显色培养基。
优选的,所述霉菌和酵母菌冷水可溶性凝胶粉末的制备:将过100~150 目标准筛后的海藻酸钠和瓜尔胶混合,得到霉菌和酵母菌冷水可溶凝胶粉末,其中海藻酸钠和瓜尔胶的质量比为4:1。
优选的,所述步骤(2)中,需氧菌的培养温度为30~35℃,培养3~5 天;霉菌和酵母菌培养温度为20~25℃,培养5~7天。
优选的,所述步骤(3)中,需氧菌的培养温度为30~35℃,培养2天;霉菌和酵母菌培养温度为20~25℃,培养2天。
与现有技术相比,本发明的有益效果如下:
菌落测试片法作为一种新型检测手段凭借质轻、便于运输和成本低等优点在食品、医学、农业等多个领域得到了广泛应用。菌落测试片是一种预先制备好的一次性培养基制品,含有微生物生长所选择性营养基质及特异脱氢酶指示剂,具有快速、方便、灵敏度高、特异性强、操作简便、便于运输、成本低等优点,有良好的研究价值和应用前景。本发明结合中国药典微生物计数法和菌落测试片的使用,最大程度减少系统适用性的改变,将微生物计数时长由7天缩短到2天。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
需氧菌总数快检方法回收率试验(试验菌包括金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌)。
用10ml的0.9%无菌氯化钠溶液分别将试验菌种(金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌)从其斜面上洗脱下来收集到无菌试管中,再用 0.9%无菌氯化钠溶液采用10倍稀释法制成试验菌悬液。菌悬液浓度为不大于100cfu/ml。
药典组:吸取1ml试验菌悬液注入无菌平皿中,倾注温度不超过45℃的胰酪大豆胨琼脂培养基,每株试验菌平行制备2个平皿。
快检组:吸取1ml试验菌悬液加至需氧菌总数测试片,每株试验菌平行制备2片。
制备好的平皿和测试片,倒置于33℃培养,平皿培养5天,测试片培养 2天,对每个平皿和测试片点计菌落数,以算术均值作为计数结果。
需氧菌总数快检方法回收率试验结果见表1
表1需氧菌总数快检方法回收率试验结果
各试验菌的回收试验(快检组平均菌落数的值与药典组平均菌落数的比值)均在0.5~2范围内,符合要求。
霉菌和酵母菌总数快检方法回收率试验(试验菌包括白色念珠菌、黑曲霉)。
用10ml的0.9%无菌氯化钠溶液分别将白色念珠菌从其斜面上洗脱下来收集到无菌试管中,再用0.9%无菌氯化钠溶液采用10倍稀释法制成试验菌悬液。黑曲霉用10ml含0.05%(ml/ml)聚山梨酯80的0.9%无菌氯化钠溶液将孢子洗脱并收集到无菌试管中,然后用含0.05%(ml/ml)聚山梨酯80的0.9%无菌氯化钠溶液采用10倍稀释法制成试验菌悬液。菌悬液浓度为不大于100cfu/ml。
药典组:吸取1ml试验菌悬液注入无菌平皿中,倾注温度不超过45℃的沙氏葡萄糖琼脂培养基,每株试验菌平行制备2个平皿。
快检组:吸取1ml试验菌悬液加至霉菌和酵母菌总数测试片,每株试验菌平行制备2片。
霉菌和酵母菌总数快检方法回收率试验结果见表2
表2霉菌和酵母菌总数快检方法回收率试验结果
各试验菌的回收试验(快检组平均菌落数的值与药典组平均菌落数的比值)均在0.5~2范围内,符合要求。
实施例2
选取非无菌药品中间体3批,分别取10g供试品,加入至90mL稀释液中,制备成1:10供试液,用同一稀释液将供试液进一步10倍系列稀释。
需氧菌总数计数:选取2至3个适宜的稀释级,每个稀释级分别取1mL 供试液加入胰酪大豆胨琼脂培养基中,平行制备2皿,置33℃培养5天;同时每个稀释级分别取1mL供试液加至需氧菌总数测试片,平行制备2片,置 33℃培养2天。培养结束后,观察菌落生长情况并计数。
霉菌和酵母菌总数计数:每个稀释级分别取1mL供试液加入沙氏葡萄糖琼脂培养基中,平行制备2皿,置23℃培养7天。同时每个稀释级分别取1mL 供试液加至霉菌和酵母菌总数测试片,平行制备2片,置23℃培养2天。培养结束后,观察菌落生长情况并计数。需氧菌总数计数结果,霉菌和酵母菌总数计数结果见表3。
表3非无菌药品中间体菌落总数计数结果
综上所述,本发明具有快速、方便、灵敏度高、特异性强、操作简便、便于运输等优点,结合中国药典微生物计数法和菌落测试片的使用,最大程度减少系统适用性的改变,将微生物计数时长由7天缩短到2天。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (1)
1.一种用于非无菌药品中间体微生物计数的快速检测方法,其特征在于:包括以下步骤:
(1)依据2020版中国药典四部1106、1107制备供试液;
(2)药典组:每个稀释级分别取1mL供试液加入胰酪大豆胨琼脂培养基中,平行制备2皿,置20~35℃培养3至7天;
(3)快检组:揭开测试片的上层膜,取供试液1ml加至测试片涂有计数显色培养基的镂空区域,使供试液均匀分布于整个培养区,将上层膜缓缓落下,避免挤压,静置30s~1min后放入恒温培养箱于20~35℃培养2天,按照2020版中国药典四部进行计数,每个稀释度平行制备两片,同时取1ml生理盐水作空白对照;
(4)判定:药典组和快检组两组试验平行进行,培养结束后,观察菌落生长情况并计数,若测试片计数结果符合规定,则判定非无菌药品中间体菌总数合格;若测试片计数结果不符合规定,或平皿法计数结果不符合规定,则判定非无菌药品中间体菌总数不合格;
其中,所述测试片包括用于测定需氧菌总数的需氧菌总数计数测试片或用于测定霉菌和酵母菌总数的霉菌和酵母菌总数计数测试片;
其中,所述需氧菌总数的需氧菌总数计数测试片由如下步骤制备而成:
将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP底层裁剪成相同尺寸;将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP底层的其中一边均留出3~5mm宽度粘合;将需氧菌冷水可溶凝胶粉末均匀地涂在PET复合膜的粘性面上;将需氧菌总数显色培养基与需氧菌冷水可溶性凝胶粉末按质量比8:1~2配制,再将得到的混合粉末均匀涂抹在PVC中层板的镂空区域内;
所述需氧菌总数显色培养基的制备:称取胰蛋白胨18~25g,大豆木瓜蛋白酶水解物5~12g,牛肉浸粉3~8g,氯化钠3~7g,葡萄糖2~6g,红四氮唑0.2~0.5g,单甘酯0.2~0.5g,混匀后得到需氧菌总数计数显色培养基;
所述需氧菌冷水可溶性凝胶粉末的制备:将经研磨后过100~150目标准筛的黄原胶和瓜尔胶混合,得到需氧菌冷水可溶凝胶粉末,其中黄原胶和瓜尔胶的质量比为7:2~3;
所述霉菌和酵母菌总数计数测试片由如下步骤制备而成:将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP底层裁剪成相同尺寸;将一面带有粘性的PET复合膜、带有镂空区域的PVC板和BOPP底层的其中一边均留出3~5mm宽度粘合;将霉菌和酵母菌冷水可溶凝胶粉末均匀地涂在PET复合膜的粘性面上;将裁圆的灭菌无纺布浸泡于霉菌和酵母菌总数计数显色培养基中10~20分钟后取出,在无菌条件下烘干,贴于PVC中层板的镂空区域内;
所述霉菌和酵母菌总数计数显色培养基的制备:称取胰蛋白胨15~25g,大豆木瓜蛋白酶水解物5~10g,葡萄糖15~20g,氯化钠2~8g,硫酸镁0.1~0.3g,硫酸铜0.1~0.3g,加入1000ml纯化水中,经121℃,15min高压灭菌后冷却至室温,再加入滤菌后的氯霉素0.1~0.5g、5-溴-4-氯-3-吲哚基-N-乙酰基-β-D-氨基半乳糖苷0.2~0.4g,制得霉菌和酵母菌总数计数显色培养基;
所述霉菌和酵母菌冷水可溶性凝胶粉末的制备:将过100~150目标准筛后的海藻酸钠和瓜尔胶混合,得到霉菌和酵母菌冷水可溶凝胶粉末,其中海藻酸钠和瓜尔胶的质量比为4:1。
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