PCR-DGGE指纹技术与分离技术结合筛选藏灵菇奶发酵过程的优势菌

【目的】获得藏灵菇发酵奶发酵过程的优势菌，并开发出纯培养发酵剂替代藏灵菇进行这类新型发酵奶的工业化生产。【方法】将PCR-DGGE指纹技术和传统培养方法结合，对藏灵菇发酵奶中微生物种群结构进行研究。【结果】DGGE指纹图谱显示混合菌群中细菌有4条条带无对应的纯菌株，而纯菌株中有2株在混合菌群的图谱上没有相应的条带。通过对150株分离菌的PCR-DGGE指纹图谱分析，获得了8组乳酸菌和5组酵母菌，进一步的序列分析和相似性比较，确立了藏灵菇发酵奶中的优势菌为肠膜明串珠菌、乳酸乳球菌、开菲尔乳杆菌、干酪乳杆菌和克鲁维酵母，单孢酵母、啤酒酵母、假丝酵母发酵后期成为优势菌。【结论】本研究建立了一种基于分子生态的微生物高效筛选技术，为藏灵菇纯培养发酵剂的开发提供了理论依据及丰富的菌种资源。

Screen of Dominating Microbial Species Isolated From Tibetan Kefir Using an Integrated Approach of PCR-DGGE and Culture-Dependent Methods

【Objective】To screen dominating microbial species isolated from Tibetan Kefir and develop pure-culture starter. 【Method】PCR-DGGE and traditional culture-depending methods were combined to examine the microbial community of Tibetan kefir. 【Result】 Four bands in the DGGE profiles of total bacterial DNA of Tibetan kefir were not recovered by cultivation. Conversely, two bacterial species did not exhibit bands in DGGE fingerprints of total bacterial DNA of Tibetan kefir. 150 strains isolated were screened and grouped by PCR-DGGE, eight lactic acid bacteria and five yeasts were obtained. Comparison of 16S rDNA (bacteria) and 26S rDNA (yeast) partial sequence similarity revealed that the dominating microbial species of Tibetan kefir were Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus kefiri, Lactobacillus casei and Kluyveromyces marxianus, Saccharomyces unisporus, Saccharomyces cerevisiae and Candida humilis were dominating during last fermentation. 【Conclusion】 An effective microbial screening technology based on molecular microbiology was established in this study, providing theoretical basis and rich microbial resources for the development of Tibetan Kefir pure-culture starter.