地衣芽孢杆菌活菌制剂芽孢数的快速检测方法研究

Research on Rapid Detection Method for Spore Counting of Viable Organism Preparation of Bacillus Licheniformis

关键词:  地衣芽孢杆菌活菌制剂  高效液相色谱法  芽孢数测定  平板计数法

Key words:  viable organism preparation of Bacillus licheniformis  HPLC  spore counting  plate counting method

摘要:

目的 利用HPLC建立地衣芽孢杆菌活菌制剂中芽孢数的快速检测方法。方法 采用HPLC，以Waters Xbridge C₁₈色谱柱（4.6 mm×250 mm，5 μm）、采用0.1%磷酸溶液-甲醇（92：8）为流动相洗脱、柱温30℃、检测波长273 nm、流速0.8 mL·min^-1，建立地衣芽孢杆菌中2，6-吡啶二羧酸（DPA）含量的测定方法。以平板计数法对相应的芽孢悬液中芽孢数量进行统计，建立DPA浓度与芽孢数之间的关系曲线。同时检测3种地衣芽孢杆菌活菌制剂（片剂、胶囊剂和颗粒剂）的芽孢数。结果 建立的检测方法稳定性、精密度、重复性、加样回收率均良好，DPA线性关系良好，线性范围为2.5~80 mg·L^-1（r=0.999），平均回收率为102.3%（n=9）；芽孢最终浓度与DPA浓度呈线性关系，芽孢最终浓度线性范围为6.5×10⁸7.8×10⁹ CFU·mL^-1（r=0.993）。同时检测3种地衣芽孢杆菌活菌制剂（片剂、胶囊剂和颗粒剂）的芽孢数，结果无显著性差异。结论 建立地衣芽孢杆菌活菌制剂中芽孢数的快速检测方法，效果好、简单、快速。

Abstract:

OBJECTIVE To establish a rapid detection method for spore counting of viable organism preparation of Bacillus licheniformis by HPLC. METHODS HPLC was adopted to determine dipicolinic acid(DPA) which was released by Bacillus licheniformis, using a Waters Xbridge C₁₈ column(4.6 mm×250 mm, 5 μm) with mobile phase consisting of 0.1% phosphoric acid-methanol(92:8), while the column temperature was maintained at 30℃, the flow rate was 0.8 mL·min^-1 and the detection wavelength was 273 nm. Plate counting method was adopted to determine spores in corresponding spore suspension in order to establish a relationship curve between the concentration of DPA and spore counting. The methods were applied to detect the spore count in three viable organism preparations of Bacillus licheniformis(tablet, capsule and granule), at the same time. RESULTS Stability, precision, repetition and recovery rates of the method established were all good. The standard curve of DPA was linear over the range of 2.5-80 mg·L^-1(r=0.999), with the average recovery rate of 102.3%(n=9). There was a linear relationship between the concentration of the spore counting and DPA with a range of spore counting concentration of 6.5×10⁸-7.8×10⁹ CFU·mL^-1(r=0.993). There were no significant differences in spore counting of three viable organism preparations of Bacillus licheniformis(tablet, capsule and granule). CONCLUSION Method of rapid detection for spore counting of viable organism preparation of Bacillus licheniformis is well worked, simple and rapid.