Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata / 中国实验方剂学杂志

ABSTRACT

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 10th 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 1×106 CFU·mL-1 at the end of fermentation. At 5 different fermentation time points, the copy numbers of Bacillus subtilis were 3.53×105, 7.56×104, 1.58×105, 1.90×106, 1.85×106 copies·g-1, the copy numbers of Paecilomyces variotii were 0, 0, 0, 3.45×107, 4.15×108 copies·g-1, the copy numbers of Byssochlamys spectabilis were 0, 0, 0, 1.04×108, 2.28×108 copies·g-1, the copy numbers of Aspergillus niger were 0, 0, 9.48×105, 1.47×106, 7.56×106 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.