Structural and Functional Changes in Prokaryotic Communities in Artificial Pit Mud during Chinese Baijiu Production

Abstract

The Chinese alcoholic beverage strong-flavor baijiu (SFB) gets its characteristic flavor during fermentation in cellars lined with pit mud. Microbes in the pit mud produce key precursors of flavor esters. The maturation time of natural pit mud of over 20 years has promoted attempts to produce artificial pit mud (APM) with a shorter maturation time. However, knowledge about the molecular basis of APM microbial dynamics and associated functional variation during SFB brewing is limited, and the role of this variability in high-quality SFB production remains poorly understood. We studied APM maturation in new cellars until the fourth brewing batch using 16S rRNA gene amplicon sequencing, quantitative PCR, metaproteomics, and metabolomics techniques. A total of 36 prokaryotic classes and 195 genera were detected. Bacilli and Clostridia dominated consistently, and the relative abundance of Bacilli decreased along with the APM maturation. Even though both amplicon sequencing and quantitative PCR showed increased abundance of Clostridia, the levels of most of the Clostridium proteins were similar in both the first- and fourth-batch APM samples. Six genera correlated with eight or more major flavor compounds in SFB samples. Functional prediction suggested that the prokaryotic communities in the fourth-batch APM samples were actively engaged in organic acid metabolism, and the detected higher concentrations of proteins and metabolites in the corresponding metabolic pathways supported the prediction. This multi-omics approach captured changes in the abundances of specific microbial species, proteins, and metabolites during APM maturation, which are of great significance for the optimization of APM culture technique. IMPORTANCE Strong-flavor baijiu (SFB) accounts for more than 70% of all Chinese liquor production. In the Chinese baijiu brewing industry, artificial pit mud (APM) has been widely used since the 1960s to construct fermentation cellars for production of high-quality SFB. To gain insights at the systems level into the mechanisms driving APM prokaryotic taxonomic and functional dynamics and into how this variation is connected with high-quality SFB production, we performed the first combined metagenomic, metaproteomic, and metabolomic analyses of this brewing microecosystem. Together, the multi-omics approach enabled us to develop a more complete picture of the changing metabolic processes occurring in APM microbial communities during high-quality SFB production, which will be helpful for further optimization of APM culture technique and improvement of SFB quality.

Microbial community changes during the mechanized production of light aromaXiaoqu baijiu

“…The diversity increased slightly after day 10, and many alcohol-tolerant microorganisms began to grow. The changes in environment and nutrition during brewing explain the changes in community species diversity [42]. The diversity index reflects the changes in the microbial community, as confirmed in the present study.…”

Mining the Factors Driving the Evolution of the Pit Mud Microbiome under the Impact of Long-Term Production of Strong-Flavor Baijiu

“…According to the national standard of strong-flavor baijiu (number GB/T 10781.1-2006) ( 5 ), the concentrations of total esters and ethyl hexanoate are set as key parameters to classify the quality grade. Both recent research and long-term production practice have highlighted the crucial roles of microbes inhabiting pit mud in the accumulation of typical flavor compounds (mainly hexanoic acid and butyric acid) of strong-flavor baijiu ( 6 – 8 ). Thus, elucidating the features of the pit mud microbial community structure and function will provide key information to understand the complex flavor formation mechanisms of strong-flavor baijiu.…”

“…In addition, the diversity and abundance of Clostridia were significantly higher in normal and high-quality pit mud than in the degraded samples, while Lactobacillus decreased with the improvement of quality ( 18 ). Accordingly, a major goal of pit mud production is to increase the levels of clostridial microbes to meet the target of hexanoic acid accumulation enhancement, which will further help improve product flavor ( 8 , 19 ). The main objective of this work was to explore the forces modulating the pit mud microbiome on the basis of the microbial community structure and metabolic characteristics analysis of different-aged samples.…”

Culturing Bacteria From Fermentation Pit Muds of Baijiu With Culturomics and Amplicon-Based Metagenomic Approaches

“…Microbial diversity in different PM has been widely revealed in recent years. Liu et al (2020) found that Clostridium and Bacillus were dominant bacterial genera in PM with high relative abundance during Chinese Baijiu production. However, there are significant differences in microbial community structure in different PM samples.…”