百药煎传统炮制过程中微生物的分离与初步鉴定及其鞣质水解能力测定

摘要

目的:揭示百药煎传统炮制过程中的微生物菌群,同时初步考察所分离菌株鞣质水解能力.方法:应用经典微生物分离纯化方法,结合形态学考察和16S rDNA或18S rDNA基因序列分析,对百药煎炮制过程中样品进行微生物的分离及菌种初步分类鉴定.采用含单宁酸的固体培养基初步筛选具有鞣质降解能力的菌株.结果:共分离获得细菌7株、酵母菌7株、丝状真菌3株.7种细菌分别为枯草芽孢杆菌、蜡样芽孢杆菌、地衣形芽孢杆菌、黄海芽孢杆菌、巨大芽孢杆菌菌株、同温层芽孢杆菌、其他芽孢杆菌;7种酵母菌分别为伯顿丝孢毕赤酵母、2株克鲁维酵母菌株、弗比恩酵母菌、奥默柯达菌、异常威克汉姆酵母菌、异常毕赤酵母菌;3株丝状真菌分别为青霉菌、卷枝毛霉菌、黑曲霉菌.除枯草芽孢杆菌、黄海芽孢杆菌、芽孢杆菌、克鲁维酵母菌HMY3、弗比恩酵母菌外,其余菌株均具有鞣质降解能力.结论:百药煎传统发酵炮制工艺涉及多种微生物共同参与混合发酵过程,初步筛选证明5株细菌、5株酵母菌和3株丝状真菌具有鞣质降解能力,这一发现为研究百药煎现代炮制工艺奠定了微生物菌种基础.%Objective:To reveal the beneficial microorganisms in processed Chinese gall leaven during their processing by traditional fermentation methods.Methods:Using classical microbial isolation and purification methods,the strains in processed Chinese gall leaven samples were isolated and purified.The preliminary taxonomy of the strains isolated was carried out by investigating the morphological properties,and analyzing and comparing their 16S rDNA or 18S rDNA gene sequences.The ability of degradating tannic acid was determined.Results:Seven species of bacteria,seven species of yeasts and three species of filamentous fungi were isolated and identified in processed Chinese gall leaven samples.The bacteria include Bacillus subtilis,Bacillus cereus,Bacillus lcheniformis,Bacillus,marisflavi,Bacillus megaterium,Bacillus stratosphericus,and Paenibacillus sp.,the yeasts were Hyphopichia burtonii,Kluyveromyces marxianus,Klayveromyces marxianus,Cyberlindnera fabianii,Kodamaea ohmeri,Wickerhamomyces anomalus,and Pichia anomala,and the fungi are Aspergillus niger,Mucor circinelloides,Penicillium spp.By determining the ability of degradating tannic acid,other strains all showed to be positive except for Bacillus subtilis,Bacillus marisflavi,Paenibacillus sp.,Kluyveromyces marxianus HMY3,Cyberlindnera fabianii.Conclusion:Many species of microorganisms exist in processed Chinese gall leaven samples during its processing,in which five species of bacteria,five species of yeasts and three species of filamentous fungi are able to degrade tannic acid,which help to reconstruct modern fermentation technology of processed Chinese gall leaven.