Department of Viticulture and Enology, University of California Davis, Davis, California, United States of America.
PLoS One. 2012;7(5):e36357. doi: 10.1371/journal.pone.0036357. Epub 2012 May 1.
While wine fermentation has long been known to involve complex microbial communities, the composition and role of bacteria other than a select set of lactic acid bacteria (LAB) has often been assumed either negligible or detrimental. This study served as a pilot study for using barcoded amplicon next-generation sequencing to profile bacterial community structure in wines and grape musts, comparing the taxonomic depth achieved by sequencing two different domains of prokaryotic 16S rDNA (V4 and V5). This study was designed to serve two goals: 1) to empirically determine the most taxonomically informative 16S rDNA target region for barcoded amplicon sequencing of wine, comparing V4 and V5 domains of bacterial 16S rDNA to terminal restriction fragment length polymorphism (TRFLP) of LAB communities; and 2) to explore the bacterial communities of wine fermentation to better understand the biodiversity of wine at a depth previously unattainable using other techniques. Analysis of amplicons from the V4 and V5 provided similar views of the bacterial communities of botrytized wine fermentations, revealing a broad diversity of low-abundance taxa not traditionally associated with wine, as well as atypical LAB communities initially detected by TRFLP. The V4 domain was determined as the more suitable read for wine ecology studies, as it provided greater taxonomic depth for profiling LAB communities. In addition, targeted enrichment was used to isolate two species of Alphaproteobacteria from a finished fermentation. Significant differences in diversity between inoculated and uninoculated samples suggest that Saccharomyces inoculation exerts selective pressure on bacterial diversity in these fermentations, most notably suppressing abundance of acetic acid bacteria. These results determine the bacterial diversity of botrytized wines to be far higher than previously realized, providing further insight into the fermentation dynamics of these wines, and demonstrate the utility of next-generation sequencing for wine ecology studies.
虽然葡萄酒发酵长期以来一直被认为涉及复杂的微生物群落，但除了一组精选的乳酸菌（LAB）之外，其他细菌的组成和作用通常被认为是微不足道的或有害的。本研究旨在使用带有条形码的扩增子下一代测序（NGS）来分析葡萄酒和葡萄汁中的细菌群落结构，比较通过对两个不同原核 16S rDNA 区域（V4 和 V5）进行测序所达到的分类深度。本研究旨在实现两个目标：1）通过比较细菌 16S rDNA 的 V4 和 V5 区域与 LAB 群落的末端限制性片段长度多态性（TRFLP），从经验上确定用于葡萄酒条形码扩增子测序的最具分类学信息量的 16S rDNA 目标区域；2）探索葡萄酒发酵过程中的细菌群落，以更好地了解使用其他技术以前无法达到的葡萄酒的生物多样性。V4 和 V5 扩增子的分析提供了酒香发酵细菌群落的相似视图，揭示了广泛的低丰度菌群多样性，这些菌群传统上与葡萄酒无关，以及最初通过 TRFLP 检测到的非典型 LAB 群落。V4 区域被确定为更适合葡萄酒生态学研究的阅读方法，因为它为分析 LAB 群落提供了更大的分类深度。此外，还使用靶向富集从已完成的发酵中分离出两种α变形菌。接种和未接种样品之间的多样性存在显著差异，这表明酿酒酵母接种对这些发酵过程中的细菌多样性施加了选择性压力，特别是抑制了醋酸菌的丰度。这些结果确定酒香葡萄酒的细菌多样性远高于以前的认识，进一步深入了解这些葡萄酒的发酵动态，并证明了下一代测序在葡萄酒生态学研究中的实用性。
