半夏曲炮制过程中微生物数量动态变化的初步分析

Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata

目的:检测半夏曲炮制过程中细菌、酵母菌、霉菌的菌落数量并定量分析其中4种优势微生物数量的动态变化,为研究半夏曲炮制机制提供实验依据。方法:参照《中华人民共和国卫生部药品标准中药成方制剂》(第十册)制备半夏曲,取半夏曲炮制过程中0,30,60,90,120 h共5个不同时间点样品,分别用选择性培养基进行细菌、霉菌、酵母菌的培养和分离纯化,并进行菌落计数;应用荧光定量聚合酶链式反应(PCR)技术对枯草芽孢杆菌Bacillus subtilis,宛氏拟青霉Paecilomyces variotii,丝衣霉菌Byssochlamys spectabilis,黑曲霉Aspergillus niger进行绝对定量,分别以枯草芽孢杆菌、宛氏拟青霉、丝衣霉菌、黑曲霉重组质粒为对照品,经倍比稀释后制作标准曲线,对半夏曲炮制至5个不同时间点(0,30,60,90,120 h)样品中的4种微生物进行定量分析。结果:半夏曲炮制过程中细菌数量少且变化平缓,而酵母菌和霉菌数量至发酵后期时迅速增加,至发酵结束时均>1×10^6CFU·m L^-1。枯草芽孢杆菌5个不同时间点样品中的拷贝数分别为3.53×10^5,7.56×10^4,1.58×10^5,1.90×10^6,1.85×10^6copies·g^-1;宛氏拟青霉的拷贝数为0,0,0,3.45×10^7,4.15×10^8copies·g^-1;丝衣霉菌的拷贝数为0,0,0,1.04×10^8,2.28×10^8copies·g^-1;黑曲霉的拷贝数为0,0,9.48×10^5,1.47×10^6,7.56×10^6copies·g^-1。结论:半夏曲炮制过程中微生物菌群变化趋势可以用4种优势微生物的动态变化来反映,霉菌在半夏曲炮制中可能起重要作用。荧光定量PCR技术具有快速灵敏、重复性好和特异性高的优点,适用于半夏曲的炮制机制研究。

Objective:To detect the colony number of bacteria,yeasts and molds in fermentation process of Pinelliae Rhizoma Fermentata(PRF),microbial flora species,and quantitatively analyze the dynamic changes of four dominant microorganisms at different fermentation time points of PRF,so as to provide experimental basis for exploring the processing mechanism of PRF.Method:According to Pharmaceutical Standard Preparation of Traditional Chinese Medicine Prescription of Ministry of Health of the People’s Republic of China(the 10^ th volume),PRF was processed.The samples at five different fermentation time points(0,30,60,90,120 h)of PRF were taken,the culturing,isolation and purification of bacteria,yeasts and molds were carried out with selective media,and the colonies were counted.Fluorescence quantitative polymerase chain reaction(PCR)technique was employed to conduct absolute quantification of Bacillus subtilis,Paecilomyces variotii,Byssochlamys spectabilis and Aspergillus niger.The recombinant plasmids of these 4 microorganisms were used as the standard substances,and the standard curves were prepared after dilution of multiple ratios,quantitative analysis was performed on these 4 microorganisms in five samples at different processing time points(0,30,60,90,120 h)of PRF.Result:During the fermentation process of PRF,the number of bacteria was low with smooth change,while molds and yeasts grew dramatically at the late stage of fermentation and reached 1×10^6 CFU·m L^-1 at the end of fermentation.At 5 different fermentation time points,the copy numbers of Bacillus subtilis were 3.53×10^5,7.56×10^4,1.58×10^5,1.90×10^6,1.85×10^6 copies·g^-1,the copy numbers of Paecilomyces variotii were 0,0,0,3.45×10^7,4.15×10^8 copies·g^-1,the copy numbers of Byssochlamys spectabilis were 0,0,0,1.04×10^8,2.28×10^8 copies·g^-1,the copy numbers of Aspergillus niger were 0,0,9.48×10^5,1.47×10^6,7.56×10^6 copies·g^-1,respectively.Conclusion:The change trend of microflora in the fermentation process of PRF can be reflected by the dynamic change of four dominant microorganisms,and molds may play an important role in the processing of PRF.Fluorescence quantitative PCR technique has the advantages of rapidity,sensitivity,good repeatability and high specificity,it is suitable for exploring processing mechanism of PRF.