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.

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.