RESUMO
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.
RESUMO
The complex starter culture for artificial pit mud (APMSC) hosts a wide variety of microbial communities that play a crucial role in Chinese strong-flavor Baijiu production. Based on its organoleptic properties, the quality of APMSC can be divided into normal and inferior quality grades. However, the relationship between the APMSC microbial community and APMSC quality is poorly understood. In this study, the bacterial community structure in normal and inferior APMSC derived from two different production batches was analyzed using denaturing gradient gel electrophoresis and Illumina MiSeq sequencing. Highly similar patterns of bacterial diversity and community structure were observed in the APMSC samples of the same quality, and a significant higher bacterial species diversity (Shannon index and Chao1) was detected in the normal compared to the inferior APMSC samples. Fifteen genera were detected in the APMSC samples, and seven (Caproiciproducens, Clostridium, Lactobacillus, Bacillus, Pediococcus, Rummeliibacillus, and Sporolactobacillus) were dominant, accounting for 92.12-99.89% of total abundance. Furthermore, the bacterial communities in the normal and inferior APMSC had significantly different structure and function. The normal APMSC was characterized by abundant Caproiciproducens and Clostridium and high caproic and butyric acid contents. In contrast, the inferior APMSC was overrepresented by Lactobacillus and Bacillus and lactic and acetic acids. This study may help clarify the key microbes sustaining APMSC ecosystem stability and functionality, and guide future improvements in APMSC production.
RESUMO
The pit mud (PM) in fermentation cellar is a complex ecosystem that hosts diverse microbial communities that contribute to the production of Chinese strong-flavor Baijiu (CSFB). However, the microbial ecology of PM, particularly the extent of their phylogenetic novelty remains poorly understood. Here we conducted Illumina MiSeq sequencing to explore the diversity and novelty patterns of PM bacterial communities from Luzhou Laojiao cellars in use for 40 and 400years. High diversity indices were found in the PM with 16 phyla and 105 genera. Interestingly, the compositions of dominant genera of the PM were significantly different than that reported previously for PM sampled from other geographic sites, suggesting greater microbial diversity of PM. The dominant genus of Caproiciproducens, a caproic acid-producing bacterium, is the first reported for Chinese Baijiu production. Our results demonstrate that the PM hosts a large number of novel taxa, with 26% of the total OTUs (operational taxonomic units) distant to cultured counterparts. The class Clostridia within Firmicutes presented the highest proportion of novel OTUs. Most novel OTUs were initially isolated from diverse environments, the most abundant of which came from Chinese Baijiu brewing ecosystems, highlighting the huge culturing gap within the PM, but at the same time suggesting the importance of these OTUs in CSFB production. The data presented in this study significantly increases the number of bacteria known to be associated with CSFB production and should help guide the future exploration of microbial resources for biotechnological applications.
