摘要
在《中华人民共和国药典》2015年版的基础上对新霉素微生物检定法进行了改进。以安全性更好的枯草芽孢杆菌(芽孢)作为微生物检定试验菌测定硫酸新霉素效价,建立了硫酸新霉素浓度与抑菌圈直径间的线性方程,对已标定的硫酸新霉素样品进行检测。结果显示,检测值与样品标示效价间无显著差异。进一步研究结果显示:枯草芽孢杆菌CMCC(B)63501的芽孢悬液在0~4℃可稳定保存6个月,期间所制双碟均可形成边缘规则清晰、准确度良好的抑菌圈;同时,一次制备的测活双碟经密封后,可在4℃冰箱中保存约14 d,期间测得的硫酸新霉素样品效价无显著差异,因此,一次测活双碟的制备可连续多次使用,适用于工业中长发酵周期的监测。枯草芽孢杆菌(芽孢)代替金黄色葡萄球菌作为新霉素微生物检定试验菌不仅提高了操作过程中的生物安全性,而且有效简化了实际操作流程,是一种简便、高效的新霉素效价检测方法,有望进一步推广和应用。
On the basis of Chinese Pharmacopoeia(2015), the microbial assay of neomycin was improved. The test organism of a pathogenic bacteria Staphylococcus aureus was replaced by the Bacillus subtilis(spore), which was generally recognized as safe(GRAS). The substitute strain was also sensitive to neomycin sulphate. A calibration curve between the standard neomycin sulphate concentration and the diameter of B. subtilis inhibition zone was constructed. Using the optimized method, some previously determined neomycin sulphate samples were tested and the results indicated that the measured neomycin contents were similar to those of the labeled ones and no significant differences were found. Further research exhibited that the spore suspension of Bacillus subtilis CMCC(B)63501 could be stably stored at 0-4 ℃ for 6 months, and the neomycin assay plates made during the period can form clear inhibition zones with similar diameter sizes. At the same time, the assay plates can be prepared at one time and stored at 4 ℃ refrigerator for about 14 days after being sealed, and during this period, the neomycin sulphate titers determined by these assay plates have no significant difference. Therefore, the assay plates prepared with B. subtilis spores can be stored and used for longer time than those with S. aureus. It is suitable for the monitoring of long fermentation cycles in industry. The B. subtilis(spore) instead of S. aureus as a test organism not only increases the operation safety, but also simplifies the actual operation procedues. It is a simple and efficient neomycin determination method, worthy of further application.
作者
张晶
岳荣
关莹
程绍国
张会图
ZHANG Jing;YUE Rong;GUAN Ying;CHENG Shaoguo;ZHANG Huitu(College of Bioengineering,Tianjin University of Science and Technology,Tianjin 300457;Tianjin Jinhui Pharmaceutical Group Co.,Ltd.,Tianjin 300380)
出处
《中国医药工业杂志》
CAS
CSCD
北大核心
2019年第11期1285-1289,1295,共6页
Chinese Journal of Pharmaceuticals
基金
国家自然科学基金(81373309)
