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Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.

ABSTRACT: Chinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-age-related changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated that prokaryotic diversity increased with cellar age until the age reached 25 years and that prokaryotic community structure changed significantly between three cellar ages (1, 10, and 25 years). Significant correlations between prokaryotic communities and environmental variables (pH, NH4(+), lactic acid, butyric acid, and caproic acid) were observed. Overall, our study results suggested that the long-term brewing operation shapes unique prokaryotic community structure and diversity as well as pit-mud chemistry. We have proposed a three-phase model to characterize the changes of pit-mud prokaryotic communities. (i) Phase I is an initial domestication period. Pit mud is characterized by abundant Lactobacillus and high lactic acid and low pH levels. (ii) Phase II is a transition period. While Lactobacillus abundance decreases dramatically, that of Bacteroidetes and methanogens increases. (iii) Phase III is a relative mature period. The prokaryotic community shows the highest diversity and capability to produce more caproic acid as a precursor for synthesis of ethyl caproate, the main flavor component in CSFL. This research provides scientific evidence to support the practical experience that old fermentation cellars produce high-quality liquor.

SUBMITTER: Tao Y

PROVIDER: S-EPMC3993157 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.

Tao Yong Y Li Jiabao J Rui Junpeng J Xu Zhancheng Z Zhou Yan Y Hu Xiaohong X Wang Xiang X Liu Menghua M Li Daping D Li Xiangzhen X

Applied and environmental microbiology 20140131 7

Chinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-age-related changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated ...[more]

PMID: 24487528

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Project description:Pit mud, a specific fermented soil, is an essential material for the fermentation of Chinese strong-flavour liquor. However, few studies to date have sought to characterize the spatial profiles of pit mud fungal communities in fermentation cellars from Chinese strong-flavour liquor distilleries. In this analysis, differences in fungal community structures and physicochemical properties in pit mud samples from different spatial positions within fermentation cellars were analyzed, revealing unique characteristic multidimensional pit mud fungal community profiles. Penicillium roqueforti, Pichia kudriavzevii, Aotearoamyces nothofagi, Penicillium robsamsonii, Alternaria arborescens, Trichosporon insectorum, Seltsamia ulmi, Trichosporon coremiiforme, Malassezia restricta were dominant in the pit mud samples form the upper cellar wall, whereas Metarhizium frigidum, Calonectria pseudoreteaudii, Penicillium clavigerum, Fusarium equiseti, Simplicillium chinense, Aspergillus intermedius, Trichosporon coremiiforme, Fusarium circinatum, Alternaria radicina, Aspergillus heterocaryoticus were predominant in the middle cellar wall. Alternaria radicina, Cladosporium chasmanthicola, Alternaria helianthiinficiens, Penicillium argentinense, Antarctomyces psychrotrophicus, and Trichosporon inkin are majorly present in the down cellar wall layer. Bipolaris axonopicola, Ramgea ozimecii, Penicillium argentinense, Calonectria queenslandica, Metarhizium robertsii, and Penicillium roqueforti were identified as the dominant fungi in pit mud samples from the cellar bottom. Additionally, Alternaria destruens and Alternaria doliconidium are present at notably high levels in all layers of pit mud samples. Moisture, pH, PO43-, acetic acid, humus, K+, Mg2+, Ca2+, butyric acid, and caproic acid levels in these different pit mud positions exhibited a rising incremental pattern from the upper wall layer to the bottom layer, whereas lactic acid levels were significantly lower in the bottom pit mud layer relative to these other layers. Moisture, pH, and NH4+-N were identified as the three most significant factors associated with fungal community composition through a redundancy analysis. Overall, these findings may offer a theoretical foundation for future efforts to improve or standardize artificial pit mud.

Project description: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.

Project description:BackgroundFamous Chinese strong-flavored liquor (CSFL) is brewed by microbial consortia in a special fermentation pit (FT). However, the fermentation process was not fully understood owing to the complicate community structure and metabolism. In this study, the process-related dynamics of microbial communities and main flavor compounds during the 70-day fermentation process were investigated in a simulated fermentation system.ResultsA three-phase model was proposed to characterize the process of the CSFL fermentation. (i) In the early fermentation period (1-23 days), glucose was produced from macromolecular carbohydrates (e.g., starch). The prokaryotic diversity decreased significantly. The Lactobacillaceae gradually predominated in the prokaryotic community. In contrast, the eukaryotic diversity rose remarkably in this stage. Thermoascus, Aspergillus, Rhizopus and unidentified Saccharomycetales were dominant eukaryotic members. (ii) In the middle fermentation period (23-48 days), glucose concentration decreased while lactate acid and ethanol increased significantly. Prokaryotic community was almost dominated by the Lactobacillus, while eukaryotic community was mainly comprised of Thermoascus, Emericella and Aspergillus. (iii) In the later fermentation period (48-70 days), the concentrations of ethyl esters, especially ethyl caproate, increased remarkably.ConclusionsThe CSFL fermentation could undergo three stages: saccharification, glycolysis and esterification. Saccharomycetales, Monascus, and Rhizopus were positively correlated to glucose concentration (P < 0.05), highlighting their important roles in the starch saccharification. The Lactobacillaceae, Bacilli, Botryotinia, Aspergillus, unidentified Pleosporales and Capnodiales contributed to the glycolysis and esterification, because they were positively correlated to most organic acids and ethyl esters (P < 0.05). Additionally, four genera, including Emericella, Suillus, Mortierella and Botryotinia, that likely played key roles in fermentation, were observed firstly. This study observed comprehensive dynamics of microbial communities during the CSFL fermentation, and it further revealed the correlations between some crucial microorganisms and flavoring chemicals (FCs). The results from this study help to design effective strategies to manipulate microbial consortia for fermentation process optimization in the CSFL brew practice.

Dataset Information

Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.

Publications

Prokaryotic communities in pit mud from different-aged cellars used for the production of Chinese strong-flavored liquor.

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