淡豆豉炮制过程中产γ-氨基丁酸微生物的筛选和鉴定
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摘要
课题组前期发现淡豆豉中存在较高含量的γ-氨基丁酸(GABA),该研究对淡豆豉炮制过程中不同时间点的样本进行产GABA微生物的筛选和鉴定,为研究淡豆豉炮制机制奠定基础。按2010年版《中国药典》制备淡豆豉,获取淡豆豉炮制过程中不同时间点的样本;用选择性培养基分别对不同时间点样本中的细菌、真菌进行培养、分离纯化,挑选优势菌种;将各优势菌分别接种在指定的液体培养基中进行培养,制备各优势菌发酵液;采用薄层色谱法对各菌株发酵液GABA进行定性初筛,对初筛中有GABA斑点的发酵液采用该实验室已建立的柱前在线衍生-高效液相色谱法进行定量检测,从优势菌中筛选产GABA的微生物;应用16S rDNA,18S rDNA序列分别对产GABA的细菌和真菌进行PCR扩增、对扩增产物进行测序,测序结果通过NCBI同源性比对、MEGA 7.0软件构建系统发育树进行分子生物学鉴定。该实验筛选并鉴定出9种产GABA微生物,分别是枯草芽孢杆菌Bacillus subtilis,屎肠球菌Enterococcus faecium,鸟肠球菌Enterococcus avium,溜曲霉Aspergillus tamarii,黄曲霉Aspergillus flavus,黑曲霉Aspergillus niger,极细支孢霉Cladosporium tenuissimum,桔青霉Penicillium citrinum,白腐菌Phanerochaete sordida。该研究首次从淡豆豉中筛选出9种产GABA微生物,多种产GABA的优势微生物在淡豆豉GABA的形成中可能起重要作用。
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.
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.
引文
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[21] 訾晓雪,曹宇,闫绍鹏,等.3种白腐菌木质纤维素酶活性及酶相关基因的TRAP标记遗传多态性[J].林业科学,2015,51(6):111.
[2] Kook M C,Cho S C,Kang,J Y,et al.Effect of gamma-aminobutyric acid produced by Lactobacillus sakei B2-16 on diet and exercise in high fat diet-induced Obese rats[J].Food Sci Biotechnol,2014,23(6):1965.
[3] Bhandage A K,Hellgren C,Jin Z,et al.Expression of GABA receptors subunits in peripheral blood mononuclear cells is gender dependent,altered in pregnancy and modified by mental health[J].Acta Physiol,2015,213(3):575.
[4] 牛丽颖,任艳青,王鑫国.淡豆豉异黄酮对增殖血管平滑肌细胞JAK2/STAT3信号转导通路的影响[J].中国药理学通报,2009,25(11):148.
[5] 任艳青,李清,刘姣,等.淡豆豉异黄酮对AngⅡ诱导大鼠血管平滑肌细胞周期的影响[J].中药药理与临床,2011,27(3):44.
[6] 冯薇,刘敏彦,李琛,等.淡豆豉化学成分及其体外促成骨细胞增殖活性研究[J].中国药学杂志,2016,51(3):203.
[7] 任佳秀,黄越燕,梁永红,等.应用柱前在线衍生-高效液相色谱技术测定淡豆豉中γ-氨基丁酸含量[J].中华中医药杂志,2018,33(9):3900.
[8] 李刚,梁永红,龙凯,等.再闷过程影响淡豆豉炮制工艺研究[J].中草药,2014,45(8):1083.
[9] 朱海针,龙凯,梁永红,等.Biolog技术监测淡豆豉发酵炮制过程中微生物种类动态变化[J].中国实验方剂学杂志,2015,21(17):14.
[10] 朱海针,谢卫华,龙凯,等.PCR-DGGE技术研究淡豆豉炮制过程中微生物菌群的动态变化[J].中草药,2017,48(9):1757.
[11] 郭佳佳,苏明声,王立元,等.半夏曲炮制过程中优势微生物的鉴定[J].中国中药杂志,2016,41(16):3027.
[12] 夏玉,郑华,林捷,等.屎肠球菌发酵特性及其功能性研究[J].食品工业科技,2014,35(12):123.
[13] 朱泉,程金龙,朱元召,等.产γ-氨基丁酸屎肠球菌的筛选及γ-氨基丁酸的定量[J].动物营养学报,2016,28(8):2504.
[14] 杨皓月.鸟肠球菌发酵的扇贝溶液中γ-氨基丁酸的富集[C].南京:“全球变化下的海洋与湖沼生态安全”学术交流会,2014.
[15] 李艳芳,郝建雄,程永强,等.黑曲霉和米曲霉发酵改善豆渣口感[J].农业工程报,2012,28(7):248.
[16] 庞宗文,李才,丁猛,等.桔青霉高产核酸酶P1的固态发酵条件研究[J].现代食品科技,2012,28(7):806.
[17] Ying G,Shi L,Yu Y,et al.Production,purification and characterization of nuclease P1 from Penicillium citrinum[J].Process Biochem,2006,41(6):1276.
[18] 齐鸿雁,庄群,张洪勋.溜曲霉固态发酵葵花盘生产蛋白饲料研究[J].应用与环境生物学报,1999,5(s1):182.
[19] Dae-Ho Kim,Seon-Hwa Kim,Soon-Wo Kwon,et al.Mycoflora of soybeans used for meju fermentation[J].Mycobiology,2013,41(2):100.
[20] Dae-Ho Kim,Sun-Hwa Kim,Soon-wo Kwon,et al.The mycobiota of air inside and outside the meju fermentation room and the origin of meju fungi[J].Mycobiology,2015,43(3):258.
[21] 訾晓雪,曹宇,闫绍鹏,等.3种白腐菌木质纤维素酶活性及酶相关基因的TRAP标记遗传多态性[J].林业科学,2015,51(6):111.