Objective:

2

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:

2

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:

2

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·mL

-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:

2

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.