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

Kai LONG; Li-yuan WANG; Jia-jia GUO; Mei-zhi WENG; Wei-hua XIE; Ming-sheng SU; Ming YANG; Xiao-mei XIE

doi:10.13422/j.cnki.syfjx.20191655

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

Pharmacy and Processing | 更新时间：2021-02-09

- Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 24, Pages: 78-83(2019)
- 作者机构：
  
  江西中医药大学，南昌　 330004
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20191655
  CLC： R22; Q815; R28; R943.1; Q93; TQ92
- Published：20 December 2019，
  
  Published Online：06 May 2019，
  
  Received：27 March 2019，
- 稿件说明：
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Kai LONG, Li-yuan WANG, Jia-jia GUO, et al. Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata. [J]. Chinese Journal of Experimental Traditional Medical Formulae 25(24):78-83(2019)
DOI：

Kai LONG, Li-yuan WANG, Jia-jia GUO, et al. Preliminary Research on Dynamic Change of Microbial Population in Fermentation Process of Pinelliae Rhizoma Fermentata. [J]. Chinese Journal of Experimental Traditional Medical Formulae 25(24):78-83(2019) DOI： 10.13422/j.cnki.syfjx.20191655.

摘要

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

CFU·mL

－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

，7.56×10

，1.58×10

，1.90×10

，1.85×10

copies·g

－1

；宛氏拟青霉的拷贝数为0，0，0，3.45×10

，4.15×10

copies·g

－1

；丝衣霉菌的拷贝数为0，0，0，1.04×10

，2.28×10

copies·g

－1

；黑曲霉的拷贝数为0，0，9.48×10

，1.47×10

，7.56×10

copies·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。

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 10

volume)

PRF was processed. The samples at five different fermentation time points (0

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

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

CFU·mL

-1

at the end of fermentation. At 5 different fermentation time points

the copy numbers of

Bacillus subtilis

were 3.53×10

7.56×10

1.58×10

1.90×10

1.85×10

copies·g

-1

the copy numbers of

Paecilomyces variotii

were 0

3.45×10

4.15×10

copies·g

-1

the copy numbers of

Byssochlamys spectabilis

were 0

1.04×10

2.28×10

copies·g

-1

the copy numbers of

Aspergillus niger

were 0

9.48×10

1.47×10

7.56×10

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.

关键词

半夏曲发酵炮制菌群变化优势菌种定量分析荧光定量聚合酶链式反应技术黑曲霉

Keywords

Pinelliae Rhizoma Fermentatafermentation processingmicroflora changesdominant microorganismsquantitative analysisfluorescence quantitative polymerase chain reaction techniqueAspergillus niger

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Related Institution

Changzhou Key Laboratory of Human Use Experience Research&Transformation of Menghe Medical School，Changzhou Hospital of Traditional Chinese Medicine， Nanjing University of Chinese Medicine

Third Grade Laboratory of TCM Preparations of National Administration of TCM，Key Laboratory of Liver and Kidney Diseases，Ministry of Education，Shanghai Key Laboratory of Traditional Chinese Clinical Medicine，Shuguang Hospital Affiliated to Shanghai University of TCM

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