Proteomic and high-throughput analysis of protein expression and microbial diversity of microbes from 30- and 300-year pit muds of Chinese Luzhou-flavor liquor

Luzhou-flavor liquor is fermented based on the metabolism of special microbial communities in pit. In this study, total proteins and DNAs of microbes from 30- and 300-year pit muds were firstly extracted. Meanwhile, an efficient approach for protein extraction with increased protein content was optimized. iTRAQ-based proteomic was then applied to investigate the aroma-forming functional protein expression of microbes from the samples. Furthermore, high-throughput sequencing of 16S rDNA was employed to reveal microbial diversity. We comparatively identified 63 proteins of aroma-forming functional microbes in these samples, and found that 59 of these proteins were highly expressed in the 300-year pit mud. Those aroma-forming functional proteins were found to be involved in methanogenesis, as well as the formation of caproic acid and butyric acid during the liquor fermentation. High-throughput sequencing revealed that the microbes most commonly found in both samples were members of phylum Firmicutes (by 97% sequence similarity), both of which, along with another common Methanobacterium, were important components of aroma-forming functional colonies in the pit muds for the brewing of Chinese liquor. The findings in this study afford us new insight into the different protein expression levels and microbial communities in two pit muds.