Metagenomics of ancient fermentation pits used for the production of chinese strong-aroma liquor.
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ABSTRACT:
SUBMITTER: Guo MY
PROVIDER: S-EPMC4208321 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Metagenomics of ancient fermentation pits used for the production of chinese strong-aroma liquor.
Guo Ming-Yi MY Huo Dan-Qun DQ Ghai Rohit R Rodriguez-Valera Francisco F Shen Cai-Hong CH Zhang Niang N Zhang Su-Yi SY Hou Chang-Jun CJ
Genome announcements 20141023 5
The complex microbiota of pit mud of solid-state fermentation reactors used for the production of Chinese liquor is responsible for producing one of the oldest distillates in the world. We apply a deep-sequencing approach to characterize the microbiota from pits that have been in use for up to 440 years. ...[more]
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Project description: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.
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Project description:As the main by-product of Chinese strong-flavor liquor, yellow water plays an important role in the formation of flavor components. Yellow water from different fermentation periods (30th day, 45th day, 60th day) was selected to analyze the aroma components by Headspace solid phase micro-extraction Gas Chromatography-Mass Spectrometry, and the microorganism community was evaluated by high-throughput sequencing technology and bioinformatics analysis of DNA. As the fermentation time was prolonged, the main flavor components significantly increased, and the amount of the common microbial population between yellow water and pit mud increased gradually. Among the common microorganisms, Lactobacillus accounted for the largest proportion, at about 56.96%. The microbes in the yellow water mainly belonged to Firmicutes. The abundance of Bacilli (the main bacteria) gradually decreased with time, at 87.60% at the 30th day down to 68.87% at the 60th day, but Clostridia gradually increased from 10.29% to 27.48%. At the genus level, some microbes increased significantly from the 30th day to 60th day, such as Caproiciproducens, which increased from 2.65% to 6.30%, and Sedimentibacter, increasing from 0.47% to 2.49%. RDA analysis indicated that the main aroma components were positively correlated with Clostridia and negatively correlated with Bacilli.
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Project description:At present, artificial pit mud (APM) is widely used in Chinese liquor-making industry and plays a particular role in the production of Chinese strong flavor liquor (CSFL). However, APM frequently ages during fermentation, thus becoming unsuitable for sustainable use due to its low-quality. The reasons behind, and results of, APM aging during the production of CSFL are not yet understood. Sequencing the V4 region of the 16S rRNA gene shows that prokaryotic diversity is significantly decreased (Shannon's diversity index, P < 0.01) and community composition is distinctly changed (from 1197 to 865 OTUs) in aging APM. On the phylum level, the increase of Firmicutes and decrease of Proteobacteria are the main consequences of APM aging during the production of CSFL. The counting of cultivatable bacteria confirmed that there was a large increase in Lactobacilli and aerobic spore-forming bacteria in aging low-quality APM (more than twofold). Unexpectedly, the total number of caproic acid-producing bacteria, mainly Clostridia, did not change significantly between the two kinds of APM. Furthermore, biochemical analysis indicates that the pH and the levels of NH4+ and K+ are decreased in aging low-quality APM (P < 0.01). The results obtained in this study support the possibility that environmental factors (pH, nutrients) induce the decrease of prokaryotic diversity, and the changed community composition influences the environmental properties. Therefore, through interfering with the cycle, APM aging can be controlled potentially by adjustment of environmental factors and/or supplementation of diminished or missed microorganisms.
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Project description:light aroma type liquor fermentation metagenome Raw sequence reads
| PRJNA1039311 | ENA
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.
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Project description:Natural microbiota plays an essential role in flavor compounds used in traditional food fermentation; however, the fluctuation in natural microbiota results in inconsistency in food quality. Thus, it is critical to reveal the core microbiota for flavor compound production and to construct a synthetic core microbiota for use in constant food fermentation. Here, we reveal the core microbiota based on their flavor production and cooccurrence performance, using Chinese light-aroma-type liquor as a model system. Five genera, Lactobacillus, Saccharomyces, Pichia, Geotrichum, and Candida, were identified to be the core microbiota. The synthetic core microbiota of these five genera presented a reproducible dynamic profile similar to that in the natural microbiota. A Monte Carlo test showed that the effects of five environmental factors (lactic acid, ethanol, and acetic acid contents, moisture, and pH) on the synthetic microbiota distribution were highly significant (P < 0.01), similar to those effects on a natural fermentation system. In addition, 77.27% of the flavor compounds produced by the synthetic core microbiota showed a similar dynamic profile (ρ > 0) with that in the natural liquor fermentation process, and the flavor profile presented a similar composition. It indicated that the synthetic core microbiota is efficient for reproducible flavor metabolism. This work established a method for identifying core microbiota and constructing a synthetic microbiota for reproducible flavor compounds. This work is of great significance for the tractable and constant production of various fermented foods.IMPORTANCE The transformation from natural fermentation to synthetic fermentation is essential in constructing a constant food fermentation process, which is the premise for stably making high-quality food. According to flavor-producing and cooccurring functions in dominant microbes, we provided a system-level approach to identify the core microbiota in Chinese light-aroma-type liquor fermentation. In addition, we successfully constructed a synthetic core microbiota to simulate the microbial community succession and flavor compound production in the in vitro system. The constructed synthetic core microbiota could not only facilitate a mechanistic understanding of the structure and function of the microbiota but also be beneficial for constructing a tractable and reproducible food fermentation process.
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Project description:pit mud of Chinese strong-flavor liquor Raw sequence reads
| PRJNA553105 | ENA
Project description:The popular distilled Chinese strong-flavour liquor (CSFL) is produced by solid fermentation in the ground pit. Microbes inhabiting in the pit mud (PM) on the walls of the fermentation pit are responsible for the production of caproic acid (CA) that determines the quality of CSFL to a large degree. However, little is known about the active microbial populations and metabolic potential of the PM microbiome. Here, we investigated the overall metabolic features of the PM microbiome and its active microbial components by combining metagenomics and MiSeq-sequencing analyses of the 16S rRNA genes from DNA and RNA (cDNA). Results showed that prokaryotes were predominant populations in the PM microbiome, accounting for 95.3% of total metagenomic reads, while eukaryotic abundance was only 1.8%. The dominant prokaryotic phyla were Firmicutes, Euryarchaeota, Bacteroidetes, Actinobacteria and Proteobacteria, accounting for 48.0%, 19.0%, 13.5%, 2.5% and 2.1% of total metagenomic reads respectively. Most genes encoding putative metabolic pathways responsible for the putative CA production via chain elongation pathway were detected. This indicated that the PM microbiome owned functional potential for synthesizing CA from ethanol or lactate. Some key genes encoding enzymes involved in hydrogenotrophic and acetoclastic methanogenesis pathways were detected in the PM metagenome, suggesting the possible occurrence of interspecies hydrogen transfer between CA-producing bacteria and methanogens. The 16S rDNA and 16S rRNA profiles showed that the Clostridial cluster IV, Lactobacillus, Caloramator, Clostridium, Sedimentibacter, Bacteroides and Porphyromonas were active populations in situ, in which Clostridial cluster IV and Clostridium were likely involved in the CA production. This study improved our understandings on the active populations and metabolic pathways of the PM microbiome involved in the CA synthesis in the CSFL fermentation.
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Project description:Many microorganisms in the environment participate in the fermentation process of Chinese liquor. However, it is unknown to what extent the environmental microbiota influences fermentation. In this study, high-throughput sequencing combined with multiphasic metabolite target analysis was applied to study the microbial succession and metabolism changes during Chinese liquor fermentation from two environments (old and new workshops). SourceTracker was applied to evaluate the contribution of environmental microbiota to fermentation. Results showed that Daqu contributed 9.10 to 27.39% of bacterial communities and 61.06 to 80.00% of fungal communities to fermentation, whereas environments (outdoor ground, indoor ground, tools, and other unknown environments) contributed 62.61 to 90.90% of bacterial communities and 20.00 to 38.94% of fungal communities to fermentation. In the old workshop, six bacterial genera (Lactobacillus [11.73% average relative abundance], Bacillus [20.78%], Pseudomonas [6.13%], Kroppenstedtia [10.99%], Weissella [16.64%], and Pantoea [3.40%]) and five fungal genera (Pichia [55.10%], Candida [1.47%], Aspergillus [10.66%], Saccharomycopsis [22.11%], and Wickerhamomyces [3.35%]) were abundant at the beginning of fermentation. However, in the new workshop, the change of environmental microbiota decreased the abundances of Bacillus (5.74%), Weissella (6.64%), Pichia (33.91%), Aspergillus (7.08%), and Wickerhamomyces (0.12%), and increased the abundances of Pseudomonas (17.04%), Kroppenstedtia (13.31%), Pantoea (11.41%), Acinetobacter (3.02%), Candida (16.47%), and Kazachstania (1.31%). Meanwhile, in the new workshop, the changes of microbial community resulted in the increase of acetic acid, lactic acid, malic acid, and ethyl acetate, and the decrease of ethyl lactate during fermentation. This study showed that the environmental microbiota was an important source of fermentation microbiota and could drive both microbial succession and metabolic profiles during liquor fermentation.IMPORTANCE Traditional solid-state fermentation of foods and beverages is mainly carried out by complex microbial communities from raw materials, starters, and the processing environments. However, it is still unclear how the environmental microbiota influences the quality of fermented foods and beverages, especially for Chinese liquors. In this study, we utilized high-throughput sequencing, microbial source tracking, and multiphasic metabolite target analysis to analyze the origins of microbiota and the metabolic profiles during liquor fermentation. This study contributes to a deeper understanding of the role of environmental microbiota during fermentation.
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