Optimization of Quantitative Determination of Bacitracin Based on Turbidimetric Method

doi:10.13560/j.cnki.biotech.bull.1985.2019-0994

Abstract

Abstract: The quantitative determination methods of bacitracin in the research and application are not standardized,and the results cannot be used for reference. In order to standardize the determination methods,the linear relationship between the logarithmic value of bacitracin concentration and OD₆₀₀ was established,and the initial concentration of indicating bacteria,the ratio of bacitracin solution to bacterial suspension and the culture time were optimized with repeatability and precision as indicators. The optimal determination conditions of bacitracin against bacteria by turbidimetric method were as follows：the initial concentration of indicating bacteria were 10⁷ CFU/mL,the ratio of bacitracin solution to bacterial suspension was 1∶9,and the culture time was 4 h. Under these conditions,the linear relationship was fine with R² above 0.99 and the repeatability was well. Furthermore,Escherichia coli and Staphylococcus aureus were selected to verify the feasibility of the method,and this method presented favorable repeatability and high precision. The results would provide reference and basis for the further application of turbidimetric method and the quantitative determination of bacitracin in the test and production process.

Key words: turbidimetric method, quantitative determination, bacitracin, antibacterial activity

WANG Zhi-xin, LIU Yang, ZHOU Jing-bo, HONG Dan, LU Lei-zhen, NING Ya-wei, JIA Ying-min. Optimization of Quantitative Determination of Bacitracin Based on Turbidimetric Method[J]. Biotechnology Bulletin, 2020, 36(5): 92-97.

References

[1] Cézard C, Silva -Pires V, Mullié C, et al.Antibacterial peptides:A review[J]. Formatex Research Center, 2011, 2011:926-937.
[2] Hisako S, Jun I, Eiichi M, et al.In vitro and in vivo activity of antimicrobial peptides synthesized based on the insect defensin[J]. Peptides, 2003, 25(1):19-27.
[3] 苏蕾, 马玉贞, 张广洲. 新型抗生素的研究进展及研发趋势探讨[J]. 齐鲁药事, 2007, 26(8):482-483.
[4] 白永强. 抗菌肽的分类及作用[J]. 北方牧业, 2014, (1):27-29.
[5] 李阳, 吴非, 蔡冬波, 等. 地衣芽胞杆菌DW2中敲除氨基酸转运蛋白基因yhdG提高杆菌肽产量[J]. 生物工程学报, 2018, 34(6):916-927.
[6] Haavik HI.Studies on the formation of bacitracin by Bacillus licheniformis:role of catabolite repression and organic acids[J]. Journal of General Microbiology, 1974, 84(2):321-326.
[7] 金光耀. 杆菌肽产品在动物饲料中的应用研究进展[J]. 畜牧与饲料科学, 2014, 35(5):39-40.
[8] 何晶晶, 朱倩倩, 许艺珊, 等. 杆菌肽研究进展及其应用[J]. 畜牧与饲料科学, 2015, 36(3):36-37.
[9] Potts AR, Psurek T, Jones C, et al.Validation of a quantitative HPLC method for bacitracin and bacitracin zinc using EDTA as a mobile-phase modifier[J]. J Pharm Biomed Anal, 2012, 70:619-623.
[10] Capitan-Vallvey L, Titos A, Checa R, et al.High-performance liquid chromatography determination of Zn-bacitracin in animal feed by post-column derivatization and fluorescence detection[J]. Journal of Chromatography, 2002, 943(2):227-234.
[11] The European Pharmacopeia Commission. The European Pharmacopeia(9. 0)[S]. Strasbourg:Council of Europe, 2017.
[12] The United States Pharmacopeia Commission. The United States Pharmacopeia(USP41)[S]. Rockville M:the United States Pharmacopeia Convention, 2018.
[13] The British Pharmacopeia Commission. The British Pharmacopeia(2017)[S]. British Pharmacopeia Commission Laboratory, 2017.
[14] 贾英民, 刘杨柳, 陈洲. 抗菌肽研究现状及其在食品安全中的应用前景[J]. 食品科学技术学报, 2017, 35(6):1-9.
[15] Wiegand I, Kai H, Hancock R.Agar and broth dilution methods to determine the minimal inhibitory concentration(MIC)of antimicrobial substances[J]. Nature Protocols, 2008, 3(2):163-175.
[16] 国家药典委员会. 中华人民共和国药典(2015版)[S]. 北京:中国医药科技出版社, 2015.
[17] Lalpuria M, Karwa V, Anantheswaran RC, et al.Modified agar diffusion bioassay for better quantification of Nisaplin[J]. Journal of Applied Microbiology, 2013, 114(3):663-671.
[18] 应茵, 楼娉婷. 比浊法测定多西环素效价含量[J]. 药学实践杂志, 2013, 31(5):368-370.
[19] 蔡姗英, 王妤, 蔡小惠. 比浊法测定复方地喹氯胺含片中短杆菌素的效价[J]. 海南医学院学报, 2007, 13(3):218-220.
[20] 杨亚莉, 胡昌勤. 中国药典2005年版微生物检定法的增修订情况及要点[J]. 中国抗生素杂志, 2005, 30(12):721-726.
[21] Berridge NJ, Barrett J.A rapid method for the turbidimetric assay of antibiotics[J]. J Gen Microbiol, 1952, 6(1-2):14-20.
[22] 张恩户, 乔慧, 王光建, 等. 微孔比浊法测定牛黄上清片的抗菌效价[J]. 中药药理与临床, 2012, 28(3):23-25.
[23] 王凯旋, 卫兰兰, 洪婷, 等. 分光光度计比浊法测定抗菌物质效价[J]. 食品与发酵工业, 2015, 41(3):209-214.
[24] 杨天赐, 彭宣宪. 微生物比浊法检定抗生素G-418效价[J]. 中国抗生素杂志, 2008, 33(2):90-92.
[25] 文晓玲, 刘兴兰, 任玺如. 比浊法测定阿奇霉素效价[J]. 中国药事, 2005, 19(6):349-350.
[26] 潘强, 徐秋阳, 潘智鹏, 等. 比浊法自动测定可利霉素片的效价[J]. 中国药品标准, 2012, 13(5):354-357.
[27] 周志敏, 陈少萍. 比浊法测定氯霉素效价的研究[J]. 临床合理用药杂志, 2010, 3(16):63-64.
[28] 佟斌, 吴兆亮, 殷昊, 等. 比浊法快速测定硫酸多黏菌素E效价[J]. 中国抗生素杂志, 2007, 3(5):300-302.