Chinese Liquor Fermentation: Identification of Key Flavor-Producing Lactobacillus spp. by Quantitative Profiling with Indigenous Internal Standards.

Identifying the functional microbes in spontaneous food fermentation is important for improving food quality. To identify the key flavor producers in Chinese liquor fermentation, we propose a novel quantitative microbiome profiling method that uses indigenous internal standards to normalize high-throughput amplicon sequencing results. We screened Lactobacillus acetotolerans and Lactobacillus jinshani as indigenous internal standards based on their high distribution frequencies and relative abundances. After determining the absolute abundance of indigenous internal standards using quantitative PCR with species-specific primers, the liquor-fermented bacterial community and its dynamics were better characterized by internal standards normalization. Based on quantitative microbiome profiling, we identified that Lactobacillus was a key flavor producer correlated with eight flavor compounds. Metatranscriptomic analysis indicated that Lactobacillus was active in transcribing genes involving the biosynthesis of flavor compounds and their precursors. This work has developed a novel and extensible absolute quantification method for microbiota that will alleviate concerns in the statistical analyses based on relative microbiome profiling, and shed insights into the function of Lactobacillus in food fermentation. It can potentially be applied to other microbial ecology studies.IMPORTANCE In this study, we developed a novel strategy using indigenous internal standards to normalize the high-throughput amplicon sequencing results. We chose two Lactobacillus species as indigenous internal standards and characterized the absolute abundance of the bacterial community. Further, we identified Lactobacillus as the key flavor producer using quantitative microbiome profiling combined with multivariate statistics and metatranscriptomic analysis. This work developed a novel strategy for absolute quantitative abundance analysis of microbiota and expanded our understanding of the role of Lactobacillus in food fermentation.