Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum / 中国中药杂志

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

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

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.

Identification of dominant microbes from traditional Chinese medicine Pinelliae Rhizoma Fermentata / 中国中药杂志

To investigate the microbial species, amount changes as well as the isolation and identification of domain strains at different fermentation time points of Pinelliae Rhizoma Fermentata, and provide basis for exploring the mechanism of Pinelliae Rhizoma Fermentata processing. Five samples were chosen at the time points (0, 18, 36, 54, 72 h) of Pinelliae Rhizoma Fermentata processing. Bacteria, mold and yeast from the samples were cultured; their colonies were counted, and the dominant strains were isolated and purified. The dominant bacteria and dominant fungi were identified by 16S rDNA and 26S rDNA sequencing respectively. The results showed that the bacteria count was low with slow and smooth changes in the fermentation process;while mold and yeast grew dramatically after 54 h culturing and reached 1×107 CFU•mL⁻¹ at the end of fermentation. Through the NCBI homology alignment and phylogenetic tree construction, the dominant bacteria were identified as Streptomyces sp., Bacillus pumilus, B. subtilis, B. aryabhattai and other Bacillus sp.; the dominant yeast was identified as Meyerozyma guilliermondii; the dominant mold were identified as Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger in the processing of Pinelliae Rhizoma Fermentata. The results indicated that multiple microbe species, especially yeast and mold, played a role in the fermentation processing of Pinelliae Rhizoma Fermentata. M. guilliermondii, P. variotii, P. variotii and A. niger and Bacillus sp. can be the crucial factors in the processing of Pinelliae Rhizoma Fermentata.

Submerged culture conditions for production of mycelial biomass and exopolysaccharides by Phellinus baumii / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the submerged culture conditions and nutritional requirments for the production of mycelial biomass and exopolysaccharides (EPS) by medicinal mushroom Phellinus baumii.</p><p><b>METHOD</b>The carbon sources, nitrogen sources, inoculum volume, initial pH and temperature were investigated based on shake flask cultures, respectively.</p><p><b>RESULT</b>The glucose was the most suitable carbon source for both mycelial biomass and EPS production, soy peptone was favorable nitrogen sources for both mycelial biomass and EPS production. The optimal inoculum volume, initial pH and temperature for both mycelial growth and EPS production were 6%, 6.0 and 28 degrees C, respectively.</p><p><b>CONCLUSION</b>The study obtained basic datas for large-scale submerged culture of P. baumii.</p>