Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter.
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ABSTRACT:
SUBMITTER: Huang Y
PROVIDER: S-EPMC5600954 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundChinese Nong-flavor (NF) liquor is continuously and stably produced by solid-state fermentation technology for 1000 years, resulting in enrichment of special microbial community and enzymes system in its starter. Based on traditional culture-dependent methods, these functional enzymes are hardly obtained. According to our previous metatranscriptomic analysis, which identifies plenty of thermostable carbohydrate-active enzymes in NF liquor starter, the aim of this study is to provide a direct and efficient way to mine these thermostable enzymes.ResultsIn present study, an alpha-amylase (NFAmy13A) gene, which showed the highest expression level of enzymes in starch degradation at high temperature stage (62 °C), was directly obtained by functional metatranscriptomics from Chinese Nong-flavor liquor starter and expressed in Pichia pastoris. NFAmy13A had a typical signal peptide and shared the highest sequence identity of 64% with α-amylase from Aspergillus niger. The recombinant enzyme of NFAmy13A showed an optimal pH at 5.0-5.5 and optimal temperature at 60 °C. NFAmy13A was activated and stabilized by Ca2+, and its half-lives at 60 and 70 °C were improved significantly from 1.5 and 0.4 h to 16 and 0.7 h, respectively, in the presence of 10 mM CaCl2. Meanwhile, Hg2+, Co2+ and SDS largely inhibited its activity. NFAmy13A showed the maximum activity on amylopectin, followed by various starches, amylose, glycogen, and pullulan, and its specificity activity on amylopectin was 200.4 U/mg. Moreover, this α-amylase efficiently hydrolyzed starches (from corn, wheat, and potato) at high concentrations up to 15 mg/ml.ConclusionsThis study provides a direct way to mine active enzymes from man-made environment of NF liquor starter, by which a fungal thermostable α-amylase (NFAmy13A) is successfully obtained. The good characteristics of NFAmy13A in degrading starch at high temperature are consistent with its pivotal role in solid-state fermentation of NF liquor brewing. This work would stimulate mining more enzymes from NF liquor starter and studying their potentially synergistic roles in NF liquor brewing, thus paving the way toward the optimization of liquor production and improvement of liquor quality in future.
| S-EPMC5822527 | biostudies-literature
Project description:Jiang-flavor (JF) daqu is a liquor starter used for production of JF baijiu, a well-known distilled liquor in China. Although a high temperature stage (70°C) is necessary for qualifying JF daqu, little is known regarding its active microbial community and functional enzymes, along with its role in generating flavor precursors for JF baijiu aroma. In this investigation, based on metatranscriptomics, fungi, such as Aspergillus and Penicillium, were identified as the most active microbial members and 230 carbohydrate-active enzymes were identified as potential saccharifying enzymes at 70°C of JF daqu. Notably, most of enzymes in identified carbohydrate and energy pathways showed lower expression levels at 70°C of JF daqu than those at the high temperature stage (62°C) of Nong-flavor (NF) daqu, indicating lowering capacities of saccharification and fermentation by high temperature stage. Moreover, many enzymes, especially those related to the degradation of aromatic compounds, were only detected with low expression levels at 70°C of JF daqu albeit not at 62°C of NF daqu, indicating enhancing capacities of generating special trace aroma compounds in JF daqu by high temperature stage. Additionally, most of enzymes related to those capacities were highly expressed at 70°C by fungal genus of Aspergillus, Coccidioides, Paracoccidioides, Penicillium, and Rasamsonia. Therefore, this study not only sheds light on the crucial functions of high temperature stage but also paves the way to improve the quality of JF baijiu and provide active community and functional enzymes for other fermentation industries.
| S-EPMC6423406 | biostudies-literature
Project description:Traditional Chinese liquor (Baijiu) solid state fermentation technology has lasted for several thousand years. The microbial communities that enrich in liquor starter are important for fermentation. However, the microbial communities are still under-characterized. In this study, 454 pyrosequencing technology was applied to comprehensively analyze the microbial diversity, function and dynamics of two most-consumed liquor starters (Jiang- and Nong-flavor) during production. In total, 315 and 83 bacterial genera and 72 and 47 fungal genera were identified in Jiang- and Nong-flavor liquor starter, respectively. The relatively high diversity was observed when the temperature increased to 70 and 62 °C for Jiang- and Nong-flavor liquor starter, respectively. Some thermophilic fungi have already been isolated. Microbial communities that might contribute to ethanol fermentation, saccharification and flavor development were identified and shown to be core communities in correlation-based network analysis. The predictively functional profile of bacterial communities showed significant difference in energy, carbohydrate and amino acid metabolism and the degradation of aromatic compounds between the two kinds of liquor starters. Here we report these liquor starters as a new functionally microbial resource, which can be used for discovering thermophilic and aerobic enzymes and for food and feed preservation.
| S-EPMC5674051 | biostudies-literature
Project description:The microbial community in the fermented pit determines the quantity and quality of light-flavor liquor. Genetic diversity and the potential functions of the microbial community are often analyzed by DNA-based omics sequencing. However, the features of the active microbial community have not been systematically studied. Here, metatranscriptomic analysis was performed to elucidate the active microbial composition, drivers, and their functions in light-flavor liquor fermentation. Bacterial genera, Lactobacillus, Streptococcus, Pediococcus, Thermotoga, and Faecalibacterium, and fungal genera, Saccharomyces, Talaromyces, Aspergillus, Clavispora, Rhizophagus, Cyberlindnera, and Wickerhamomyces, were the dominant active microorganisms during the fermentation process. Additionally, they dominated the three-stage fermentation successively. Redundancy analysis showed that pH, ethanol, moisture, and starch were the main driving forces of microbial succession. Among the genes for the respective carbohydrate-active enzyme families, those for the glycoside hydrolase family 23, the glycosyltransferase family 2, the carbohydrate-binding module family 50, the polysaccharide lyase family 4, the auxiliary activity family 1, and the carbohydrate esterase family 9 showed the highest expression level. Additionally, the highly expressed enzymes and their contributed microorganisms were found in the key KEGG pathways, including carbohydrate metabolism, energy metabolism, lipid metabolism, and amino acid metabolism. Based on these data, a functional model of carbohydrate hydrolysis, ethanol production, and flavor generation were proposed. Taken together, Saccharomyces, Lactobacillus, Wickerhamomyces, Pediococcus, Candida, and Faecalibacterium were suggested as the core active microorganisms. Overall, our findings provide new insights into the composition, drivers, and functions of the active microorganisms, which is crucial for improving the quality of light-flavor liquor. IMPORTANCE There is an urgent need for discovering the diversity and functions of the active microbial community in solid-state fermentation, especially in the pit of Chinese distilled liquor fermentation. Although the genetic composition of the microbial community has been clarified frequently by DNA-based sequencing, the composition and functions of the active microbial community have not been systematically revealed so far. Therefore, analysis of RNA-based data is crucial for discovering the functional microbial community. In this study, we employed metatranscriptomic analysis to elucidate the active microbial composition, successive drivers, and their functions in light-flavor liquor fermentation. The strategy can be broadly useful for discovering the active microbial community and exploring their functions in other types of flavor distilled liquor or other ecosystems. This study provides new insights into the understanding of the active microbial community composition and its functions.
| S-EPMC9241730 | biostudies-literature
Project description:The microbial diversity and the community succession in the fermenting cover lees of Chinese Luzhou-flavor liquor were investigated by small-subunit rRNA (SSU rRNA) culture independent method. All sequences retrieved from the 1, 7 and 60 days fermented cover lees were respectively assigned into the genera of Streptococcus, Acetobacter, Arthrobacter, Bacillus, Staphylococcus, Serratia, Nocardia, Methanoculleus, Clostridium, Aneurinibacillus, Corynebacterium, Lactobacillus, Microbacterium, Trichosporon, Saccharomycopsis, Sagenomella, Talaromyces, Eurotium, Issatchenkia, Zygosaccharomyces, Saccharomyces and TM7 phylum. The fungal Issatchenkia, Saccharomycopsis and Talaromyces and the bacteria Staphylococcus and Lactobacillus were most abundant in the 1 day fermented cover lees, the fungal Issatchenkia, Saccharomyces and Talaromyces and the bacteria Bacillus and Streptococcus were dominant in the 7 days cover lees, the archaea Methanoculleus and the fungal Eurotium and Talaromyces were prevalent in the 60 days cover lees. When the microbial community profiles in three samples were compared at species level, the prokaryotic community similarity coefficient was from 0.4042 to 0.5703 and descended to 0.2222, and that of eukaryotic community was from 0.3000 to 0.6000 and followed to 0.5215. These results suggested that microbial diversity variability and community succession have happened in the cover lees associated with fermentation proceeding and such variability and succession respond for the appearance of some unique flavor of Luzhou-flavor liquor.
| S-EPMC3779294 | biostudies-literature
Project description:Daqu, an important fermentation starter for the production of Chinese liquor, as used in the current study included traditional Daqu and fortified Daqu inoculated with Bacillus velezensis and Bacillus subtilis. To evaluate the effect of fortified Daqu on strong-flavor liquor production, the differences of microbial communities among three inoculation patterns of fermented grains (FG) were analyzed by the Illumina MiSeq platform. A higher relative abundance of dominant genera including Bacillus, Lactococcus, Aspergillus, and Candida, and lower relative abundance of Lactobacillus, were observed in FG50, in which mixed Daqu (traditional: fortified Daqu = 1: 1, w/w, 50% fortified Daqu) was used as the starter. Then, volatile compounds of their distillations were also examined by HS-SPME-GC-MS. The results showed that the contents of skeleton flavor components, mainly including important esters and aromatic compounds, were higher in the corresponding liquor L50, which distillated from FG50. Moreover, most esters mainly positively correlated with Lactobacillus and Candida in the bottom layer of FG fermented with 50% fortified Daqu (FG50-B). Aromatic compounds were strongly positively correlated with Bacillus and Aspergillus in the middle layer of FG with 50% fortified Daqu used (FG50-M). In particular, hexyl hexanoate showed a positive correlation with higher abundances of Ruminococcus in the FG with addition of 100% fortified Daqu (FG100). This study observed microbial compositions in the FG with fortified Daqu addition, and it further revealed the correlations between pivotal microbes and main flavor compounds. These results may help to develop effective strategies to regulate microbes for the brewing process and further improve the flavors of Chinese liquor.
| S-EPMC6361764 | biostudies-literature
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.
| S-EPMC5795089 | biostudies-literature
Project description:Selection of a starter culture with excellent viability and metabolic activity is important for inoculated fermentation of traditional food. To obtain a suitable starter culture for making Chinese sesame-flavored liquor, the yeast and bacterium community structures were investigated during spontaneous and solid-state fermentations of this type of liquor. Five dominant species in spontaneous fermentation were identified: Saccharomyces cerevisiae, Pichia membranaefaciens, Issatchenkia orientalis, Bacillus licheniformis, and Bacillus amyloliquefaciens. The metabolic activity of each species in mixed and inoculated fermentations of liquor was investigated in 14 different cocultures that used different combinations of these species. The relationships between the microbial species and volatile metabolites were analyzed by partial least-squares (PLS) regression analysis. We found that S. cerevisiae was positively correlated to nonanal, and B. licheniformis was positively associated with 2,3-butanediol, isobutyric acid, guaiacol, and 4-vinyl guaiacol, while I. orientalis was positively correlated to butyric acid, isovaleric acid, hexanoic acid, and 2,3-butanediol. These three species are excellent flavor producers for Chinese liquor. Although P. membranaefaciens and B. amyloliquefaciens were not efficient flavor producers, the addition of them alleviated competition among the other three species and altered their growth rates and flavor production. As a result, the coculture of all five dominant species produced the largest amount of flavor compounds. The result indicates that flavor producers and microbial interaction regulators are important for inoculated fermentation of Chinese sesame-flavored liquor.
| S-EPMC4068675 | biostudies-literature
Project description:pit mud of Chinese strong-flavor liquor Raw sequence reads
| PRJNA553105 | ENA
Project description:BackgroundDaqu is the most important fermentation starter for Chinese liquor, with large number of microbes and enzymes being openly enriched in the Daqu system over thousands of years. However, only a few enzymes have been analyzed with crude protein for total liquefying power and saccharifying power of Daqu. Therefore, the complex enzymatic system present in Daqu has not been completely characterized. Moreover, their pivotal and complicated functions in Daqu are completely unknown.ResultsIn this study, a novel α-amylase NFAmy13B, from GH13_5 subfamily (according to the Carbohydrate-Active enZYmes Database, CAZy) was successfully heterologous expressed by Escherichia coli from Chinese Nong-flavor (NF) Daqu. It exhibited high stability ranging from pH 5.5 to 12.5, and higher specific activity, compared to other GH13_5 fungal α-amylases. Moreover, NFAmy13B did not show activity loss and retained 96% residual activity after pre-incubation at pH 11 for 21 h and pH 12 for 10 h, respectively. Additionally, 1.25 mM Ca2+ significantly improved its thermostability. NFAmy13B showed a synergistic effect on degrading wheat starch with NFAmy13A (GH13_1), another α-amylase from Daqu. Both enzymes could cleave maltotetraose and maltopentaose in same degradation pattern, and only NFAmy13A could efficiently degrade maltotriose. Moreover, NFAmy13B showed higher catalytic efficiency on long-chain starch, while NFAmy13A had higher catalytic efficiency on short-chain maltooligosaccharides. Their different catalytic efficiencies on starch and maltooligosaccharides may be caused by their discrepant substrate-binding region.ConclusionsThis study mined a novel GH13_5 fungal α-amylase (NFAmy13B) with outstanding alkali resistance from Nong-flavor (NF) Daqu. Furthermore, its synergistic effect with NFAmy13A (GH13_1) on hydrolyzing wheat starch was confirmed, and their possible contribution in NF Daqu was also speculated. Thus, we not only provide a candidate α-amylase for industry, but also a useful strategy for further studying the interactions in the complex enzyme system of Daqu.
| S-EPMC8028695 | biostudies-literature