Research On Method Of Rapid Detection For Spore Counting Of Viable Organism Preparation Of Bacillus Licheniformis

Background:Viable organism preparation of Bacillus licheniformis is a kind of a mi croecol ogi cal agent,which has the function of enhancing human immunity and preventing diseases.In recent years,it has been widely used in clinical treatment.The key to the quality control of a microecologicalagent is to evaluate the safety and effectiveness of the products.Spore count is an important index of the performance of bacillus.The effect will be exerted with high spore count.The traditional method of detecting spore count of viable organism preparation of Bacillus licheniformis has complicated operation and long detection period,which seriously lags behind the production processObjective:By exploring the spore specific substance DPA extraction method and detection method,HPLC method for spore detection was established.The feasibility of detecting spore by FCM method was preliminarily exploredMethods:(1)The traditional plate counting method was used to count the spores of Bacillus licheniformis viable organism powder heated at different temperature for 10 min and heated at 80℃ water bath for different time.This method determined the optimal conditions for the extraction of spores;(2)High performance liquid chromatography(HPLC)was used to determine the spore specific substance—DPA,with the filter of octyl silane bonded silica gel,the mobile phase of 0.1%phosphoric acid(pH3.0)-methanol(92:8),the detection wavelength of 273 nm,the flow rate of 0.8 mL·min-1,the injecting sample of 20 μL,and the column temperature of 30℃.DPA can be quantified by this method;(3)The HPLC method was used to detect the DPA content in spores obtained by different treatment methods,so as to determine the optimal method for DPA extraction from spores;(4)Spore suspensions with different concentrations were prepared,spores were counted by plate counting method,spore suspensions were treated according to the above optimal method,DPA content was detected by HPLC method,and the relationship curve between spore count and DPA content was established;(5)The spore count of 9 batches of Bacillus licheniformis preparations of 3 dosage forms(capsule,tablet and granule)were detected by plate counting method,HPLC and FCM,respectively,to judge the feasibility of the newly established methodsResults:(1)When viable organism preparation of Bacillus licheniformis was treated at 80℃ for 30 min,the count of spore extracted was the highest;(2)Spore specific substance DPA could be quantified by high performance liquid chromatography.The results showed that DPA content in the range of 2.5 mg·L-1-80 mg·L-1 had a good linear relationship with the peak area;(3)After the spore suspension was sterilized at 121℃for 15 min,0.5 ml DTT solution was added,and 30 min of water bath at 65℃,the DPA could be extracted to the maximum;(4)The DPA concentration ranged from 7-77 mg·L-1,and the final spores concentration ranged from 6.5×108-7.8×109 CFU·mL-1,showing a good linear relationship;(5)The spores number of 9 batches of viable organism preparation of Bacillus licheniformis of 3 dosage forms(capsule,tablet and granule)were detected by plate counting method,HPLC and FCM,respectively.There was no significant difference(P>0.05).Conclusion:The HPLC and FCM method established in this study can quickly detect the spore count in the viable organism preparation of Bacillus licheniformis.The methods are accurate and reliable,and the spore number can be detected synchronously in production to shorten the production cycle.