本源乳酸菌对东北酸菜发酵微生物区系影响的分子解析

To analyze effects of epiphytic lactic acid bacteria on microflora during Chinese sauerkraut (suan-cai) fermentation, we investigate the microbial dynamics of the spontaneous suan-cai fermentation firstly. Cloning libraries are used to detect dynamics of lactic acid bacteria (LAB), bacteria and fungi. According to above results, culture-dependent method is used to isolate the LAB. Classification and identification of the prodominant LAB are be carried out based on morphological, physiochemical characteristics and sequencing. Combined with the results of volatile products from the liquid fermentation, the typical homofermentative and heterofermentative LAB (ho-LAB and he-LAB) are selected. The pure ho-LAB or/and he-LAB strain will be inoculated for the suan-cai fermentation. Besides the analyses of microbial dynamics during the fermentation by denaturing gradient gel electrophoresis, real-time quantitative PCR is used to track the inoculated LAB. At the same time, biochemical indexes of the fermented system are investigated. From the whole results, we want to understand the effects of the ho-LAB and he-LAB on the microflora of suan-cai fermentation. We expect these results will provide basic data for accelerating suan-cai industrialization and selecting the effective inoculant for the suan-cai fermentation.

为解析不同发酵类型本源乳酸菌对东北酸菜发酵微生物区系演替的影响，本立项从东北酸菜传统自然发酵入手，利用克隆文库分析自然发酵微生物群体中乳酸菌、一般细菌和真菌的动态及组成；以上述结果为基础，利用传统培养方法对东北酸菜自然发酵过程中的乳酸菌进行分离，运用形态学、生理生化反应与DNA测序相结合对优势乳酸菌进行分类及鉴定，结合液体发酵挥发性产物分析结果筛选出典型纯种同型发酵乳酸菌和异型发酵乳酸菌；利用获得的纯种同型发酵乳酸菌和异型发酵乳酸菌对东北酸菜原料进行人工接种发酵，在DGGE分析微生物区系动态及组成基础上，采用实时定量PCR方法对人工接种的乳酸菌进行追踪，结合发酵体系的生化指标分析结果，探讨接种不同发酵类型本源乳酸菌影响东北酸菜发酵进程的作用机制。研究可望在丰富东北酸菜益生菌资源的基础上，深入解析东北酸菜的微生物学发酵机理，为筛选优质高效的益生发酵剂奠定基础，以推动东北酸菜产业的规模化发展。

为深入系统了解我国东北酸菜发酵微生物区系，本项目以东北酸菜为研究对象，利用克隆文库方法研究自然发酵过程，结果表明在东北酸菜自然发酵过程中主要的细菌包括Acinetobacter sp., Pseudomonas sp., Klebsiella sp., Citrobacter sp., Beta-proteobacterium., Lactobacillus curvatus, L. plantarum 和 L. oligofermentans，Leuconostoc mesenteroides，乳酸菌为发酵的主体。真菌包括Cladosporium sp., Candida sake, Cystofiloba infirmominiatum, Tilletiopsis washingtonensis, Stramenopile sp.和soil fungus。基于上述研究结果，分离获得L. curvatus和Le.mesenteroides两种优势同型发酵乳酸菌和异型发酵乳酸菌。以其为接种剂，接入到酸菜发酵过程中，结果表明在接种处理中，Lactobacillus, Pseudomonas, 和Leuconostoc在整个发酵过程中均可检测到，Lactobacillus是主要的微生物属。定量PCR结果表明接种后的两菌可以在发酵体系内有效定殖。发酵18d内L. curvatus的DNA量迅速增加到44.7%。从发酵的12~30d内，只接种L. curvatus处理中的DNA的量是最高的。Ln. mesenteroides DNA 量随着其接种比例增加而显著增加。本研究结果不仅丰富了我国酸菜发酵微生物区系方面研究成果，而且使人们对东北酸菜发酵主体微生物有了全面、深入系统的认识。对于构建高效稳定的乳酸菌接种剂、缩短酸菜发酵周期、控制酸菜发酵质量具有重要作用。