Construction of Synthetic Microbiota for Reproducible Flavor Compound Metabolism in Chinese Light-Aroma-Type Liquor Produced by Solid-State Fermentation.
ABSTRACT: 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) 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.
Project description:Fermentation microbiota is specific microorganisms that generate different types of metabolites in many productions. In traditional solid-state fermentation, the structural composition and functional capacity of the core microbiota determine the quality and quantity of products. As a typical example of food fermentation, Chinese Maotai-flavor liquor production involves a complex of various microorganisms and a wide variety of metabolites. However, the microbial succession and functional shift of the core microbiota in this traditional food fermentation remain unclear. Here, high-throughput amplicons (16S rRNA gene amplicon sequencing and internal transcribed space amplicon sequencing) and metatranscriptomics sequencing technologies were combined to reveal the structure and function of the core microbiota in Chinese soy sauce aroma type liquor production. In addition, ultra-performance liquid chromatography and headspace-solid phase microextraction-gas chromatography-mass spectrometry were employed to provide qualitative and quantitative analysis of the major flavor metabolites. A total of 10 fungal and 11 bacterial genera were identified as the core microbiota. In addition, metatranscriptomic analysis revealed pyruvate metabolism in yeasts (genera Pichia, Schizosaccharomyces, Saccharomyces, and Zygosaccharomyces) and lactic acid bacteria (genus Lactobacillus) classified into two stages in the production of flavor components. Stage I involved high-level alcohol (ethanol) production, with the genus Schizosaccharomyces serving as the core functional microorganism. Stage II involved high-level acid (lactic acid and acetic acid) production, with the genus Lactobacillus serving as the core functional microorganism. The functional shift from the genus Schizosaccharomyces to the genus Lactobacillus drives flavor component conversion from alcohol (ethanol) to acid (lactic acid and acetic acid) in Chinese Maotai-flavor liquor production. Our findings provide insight into the effects of the core functional microbiota in soy sauce aroma type liquor production and the characteristics of the fermentation microbiota under different environmental conditions.
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.
Project description:Identifying the functional microbes in spontaneous food fermentation is important for improving food quality. To identify the key flavor producers in Chinese liquor fermentation, we propose a novel quantitative microbiome profiling method that uses indigenous internal standards to normalize high-throughput amplicon sequencing results. We screened <i>Lactobacillus acetotolerans</i> and <i>Lactobacillus jinshani</i> as indigenous internal standards based on their high distribution frequencies and relative abundances. After determining the absolute abundance of indigenous internal standards using quantitative PCR with species-specific primers, the liquor-fermented bacterial community and its dynamics were better characterized by internal standards normalization. Based on quantitative microbiome profiling, we identified that <i>Lactobacillus</i> was a key flavor producer correlated with eight flavor compounds. Metatranscriptomic analysis indicated that <i>Lactobacillus</i> was active in transcribing genes involving the biosynthesis of flavor compounds and their precursors. This work has developed a novel and extensible absolute quantification method for microbiota that will alleviate concerns in the statistical analyses based on relative microbiome profiling, and shed insights into the function of <i>Lactobacillus</i> in food fermentation. It can potentially be applied to other microbial ecology studies.<b>IMPORTANCE</b> In this study, we developed a novel strategy using indigenous internal standards to normalize the high-throughput amplicon sequencing results. We chose two <i>Lactobacillus</i> species as indigenous internal standards and characterized the absolute abundance of the bacterial community. Further, we identified <i>Lactobacillus</i> as the key flavor producer using quantitative microbiome profiling combined with multivariate statistics and metatranscriptomic analysis. This work developed a novel strategy for absolute quantitative abundance analysis of microbiota and expanded our understanding of the role of <i>Lactobacillus</i> in food fermentation.
Project description:Douchi is a type of traditional Chinese flavoring food that has been used for thousands of years and is produced by multispecies solid-state fermentation. However, the correlation between the flavor, the microbiota, and the functional core microbiota in Aspergillus-type douchi fermentation remains unclear. In this study, Illumina MiSeq sequencing and chromatography were used to investigate the bacterial community and flavor components in Aspergillus-type douchi fermentation. The dominant phyla were Firmicutes, Proteobacteria, and Actinobacteria, and the dominant genera were Weissella, Bacillus, Anaerosalibacter, Lactobacillus, Staphylococcus, and Enterococcus. A total of 58 flavor components were detected during fermentation, including two alcohols, 14 esters, five pyrazines, three alkanes, four aldehydes, three phenols, six acids, and five other compounds. Bidirectional orthogonal partial least square modeling showed that Corynebacterium_1, Lactococcus, Atopostipes, Peptostreptococcus, norank_o__AKYG1722, Truepera, Gulosibacter, norank_f__Actinomycetaceae, and unclassified_f__Rhodobacteraceae are the functional core microbiota responsible for the formation of the flavor components during douchi fermentation. This is the first study to investigate the functional core microbiota in douchi fermentation using Illumina MiSeq sequencing and chromatographic techniques. Our findings extend our understanding of the relationships between flavor, the microbiota, and the functional core microbiota during Aspergillus-type douchi fermentation.
Project description:Zygosaccharomyces bailii is a common yeast in various food fermentations. Understanding the metabolic properties and genetic mechanisms of Z. bailii is important for its industrial applications. Fermentation characteristics of Z. bailii MT15 from Chinese Maotai-flavor liquor fermentation were studied. Z. bailii MT15 produced various flavor compounds, including 19 alcohols, six acids, three esters, three ketones, and two aldehydes. Moreover, production of acids and aldehydes were increased by 110 and 41%, respectively, at 37°C (the maximum temperature in liquor fermentation) compared with that at 30°C, indicating its excellent flavor productivity. Z. bailii MT15 is a diploid with genome size of 20.19 Mb. Comparative transcriptome analysis revealed that 12 genes related to amino acid transport were significantly up-regulated (2.41- to 5.11-fold) at 37°C. Moreover, genes ARO8, ARO9, and ALDH4 involved in amino acid metabolism also showed higher expression levels (>1.71-fold) at 37°C. Increased substrate supply and a vigorous metabolism might be beneficial for the increased production of acids and aldehydes at 37°C. This work revealed the potential contribution of Z. bailii to various flavor compounds in food fermentation, and produced insights into the metabolic mechanisms of Z. bailii in flavor production.
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.
Project description:Xiaoqu is a traditional fermentation starter that is used for Chinese liquor production. Although microorganisms in the starters are closely associated with the quality and flavor of liquor, knowledge of the microbiota in xiaoqu is still far from complete, let alone the starters produced by new processes. Here, Illumina MiSeq high-throughput sequencing was applied to study bacterial composition in three types of xiaoqu used in Cantonese soybean-flavor (Chi-flavor) liquor, namely two traditional starters (Jiu Bing and Bing Wan) and a Round-Koji-maker starter (San qu) produced by the automatic starter-making disk machine. The results showed bacterial diversity in traditional starters was similar and higher than that in the Round-Koji-maker starter. Lactobacillus and Pediococcus were the dominant genera in all starters, while other different dominant genera also existed in different starters, which were Weissella, Acetobacter, and Gluconobacter for Jiu Bing, Weissella for Bing Wan, and Bacillus, Acetobacter, Acinetobacter and Klebsiella for San qu, respectively. Meanwhile, Cytophagaceae, one particular microbial family, and some pathogens including Klebsiella, Cronobacter, and Enterobacter were also found in San qu, indicating the automatic starter-making disk machine should be ameliorated before applied into industrial production. These results enriched our knowledge on xiaoqu-related microorganisms and might be helpful in industrial Chi-flavor liquor production and the development of fermentation technology.
Project description:Bacillus licheniformis CGMCC3963 is an important mao-tai flavor-producing strain. It was isolated from the starter (Daqu) of a Chinese mao-tai-flavor liquor fermentation process with solid-state fermentation. We report its genome of 4,525,096 bp here. Many potential insertion genes that are responsible for the unique properties of B. licheniformis CGMCC3963 in mao-tai-flavor liquor production were identified.
Project description:Microbial sulfur metabolism plays crucial roles in various food and alcoholic beverage fermentations. 3-(Methylthio)-1-propanol and dimethyl disulfide are important sulfur compounds in fermented foods and alcoholic beverages. Here, we studied the dynamics of these two compounds during spontaneous Chinese liquor fermentation. The two compounds reached the maximum concentration at day 10 and the maximum production rate at day 3. Metatranscriptomic analysis at days 3 and 10 revealed a total of 354 metabolically active microorganisms. Saccharomyces and Lactobacillus were identified as core microbiota critical for sulfur compound production based on both the transcript abundances of the principal genes and the distribution frequencies of 31 enzymes involved in sulfur metabolism. Saccharomyces transcribed genes encoding 23 enzymes related to the generation of 3-(methylthio)-1-propanol and dimethyl disulfide, and Lactobacillus was active in the methyl cycle, which recycles methionine, the precursor of the two sulfur compounds. Furthermore, the sulfur metabolism-related characteristics of two representative species were studied in coculture during a simulated fermentation. Saccharomyces cerevisiae JZ109 produced 158.4 μg/liter 3-(methylthio)-1-propanol and 58.5 μg/liter dimethyl disulfide in monoculture, whereas Lactobacillus buchneri JZ-JN-2017 could not produce these two compounds in monoculture. Their coculture significantly enhanced the generation of 3-(methylthio)-1-propanol (350.0 μg/liter) and dimethyl disulfide (123.8 μg/liter). In addition, coculture significantly enhanced the gene transcriptions (fold change, 1.5 to ∼55.0) that convert methionine to these two compounds in S. cerevisiae and in the methyl cycle of L. buchneri This study reveals a novel synergistic effect between members of the core microbiota in the production of sulfur compounds via methionine recycling in spontaneous Chinese liquor fermentation.IMPORTANCE Sulfur compounds play a crucial role in the aroma quality of various fermented foods and alcoholic beverages. However, it is unclear how these compounds are produced by microbes during their spontaneous fermentations. Here, we identified the core microbiota (Saccharomyces and Lactobacillus) associated with sulfur metabolism by determining both transcript abundance and distribution frequency of each genus in spontaneous Chinese liquor fermentation. This study provides a system-level analysis of sulfur metabolism by the metatranscriptomic analysis and culture-dependent methods. It sheds new light on how the metabolic behavior of the microbiota contributes to the liquor aroma quality. Furthermore, this work reveals a novel synergistic effect between Saccharomyces and Lactobacillus in the production of sulfur compounds, in which Lactobacillus regenerates the precursor methionine for sulfur compound production by Saccharomyces Our findings can contribute to the enhancement of aroma characteristics in Chinese liquor and open new avenues for improving various food and alcoholic beverage fermentation processes.
Project description:Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus, and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids, and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system, and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar.