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Importance: Baijiu is a typical example of how humans employ microorganisms to convert grains into new flavors. Mud cellars are used as the fermentation vessel for strong-flavor Baijiu (SFB) to complete the decomposition process of grains. The typical flavor of SFB is mainly attributed to the metabolites of the pit mud microbiome. China has a large number of SFB-producing regions. Previous research revealed the temporal profiles of the pit mud microbiome in different geographical regions. However, each single independent study rarely yields a thorough understanding of the pit mud ecosystem. Will the pit mud microbial communities in different production regions exhibit similar succession patterns and structures under the impact of the brewing environment? Hence, we conducted research in pit mud microbial biogeography to uncover the impact of specific environment on the microbial community over a long time scale., Competing Interests: The authors declare no conflict of interest.

As the saccharifying and fermentative agent, medium-temperature Daqu (MT-Daqu) plays an irreplaceable role in the production of strong-flavor Baijiu. Numerous studies have focused on the microbial community structure and potential functional microorganisms, however, little is known about the succession of active microbial community and the formation mechanism of community function during MT-Daqu fermentation. In this study, we presented an integrated analysis of metagenomics, metatranscriptomics, and metabonomics covering the whole fermentation process of MT-Daqu to reveal the active microorganisms and their participations in metabolic networks. The results showed that dynamic of metabolites were time-specific, and the metabolites and co-expressed active unigenes were further classified into four clusters according to their accumulation patterns, with members within each cluster displaying a uniform and clear pattern of abundance across fermentation. Based on KEGG enrichment analysis in co-expression clusters and succession of active microbial community, we revealed that Limosilactobacillus, Staphylococcus, Pichia, Rhizopus, and Lichtheimia were metabolically active members at the early stage, and their metabolic activities were conducive to releasing abundant energy to drive multiple basal metabolisms such as carbohydrates and amino acids. Thereafter, during the high temperature period and at the end of fermentation, multiple heat-resistant filamentous fungi were transcriptionally active populations, and they acted as both the saccharifying agents and flavor compound producers, especially aromatic compounds, suggesting their crucial contribution to enzymatic activity and aroma of mature MT-Daqu. Our findings revealed the succession and metabolic functions of the active microbial community, providing a deeper understanding of their contribution to MT-Daqu ecosystem., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Clostridium inhabiting pit mud (PM) is one of the important bacterial populations for synthesizing flavor compounds of Chinese strong-flavor baijiu. The long-term cereal fermentation with sorghum as the main raw material creates an environment rich in starch, ethanol, and organic acids (mainly lactic acid). However, the genetic factors underpinning Clostridium 's adaptation to PM remain poorly understood. Here, we performed comparative genomic analysis between 30 pit mud-associated (PMA) and 100 non-pit mud-associated (NPMA) Clostridium strains. Comparison analysis of the enrichment of KEGG pathways between PMA and NPMA Clostridium strains showed two-component system, flagellar assembly, and bacterial chemotaxis pathways related to environmental adaptation were enriched in PMA strains. The number of genes encoding alcohol dehydrogenase and l-lactate dehydrogenase in PMA Clostridium strains was significantly higher than that in NPMA, which is helpful for them to adapt to the ethanol- and lactic acid-rich environment. The analysis of carbohydrate-active enzymes demonstrated that glycoside hydrolases (GHs) was the most abundant family in all Clostridium strains, and genes encoding GH4 and GH13, involved in starch and sucrose metabolism, were enriched in PMA Clostridium . Horizontal gene transfer analysis revealed that multiple genes encoding the enzymes involved in carbohydrate and amino acid metabolism were transferred from Bacillus to Clostridium in pit mud. Most of the PMA Clostridium strains had good potential for butyric acid synthesis from ethanol, lactic acid, and starch. Collectively, this study furthers our understanding of the habitat adaptation and metabolic potential of PMA Clostridium strains. IMPORTANCE Pit mud is a typical artificial ecosystem for Chinese liquor production. Clostridium inhabiting pit mud plays essential roles in the flavor formation of strong-flavor baijiu. The relative abundance of Clostridium increased with pit mud quality, further influencing the quality of baijiu. So far, the ecological adaptation of Clostridium to a pit mud-associated lifestyle is largely unknown. Here, comparative genomic analysis of pit mud-associated (PMA) and non-pit mud-associated (NPMA) Clostridium strains was performed. We found genes related to the metabolism of starch, ethanol, and lactic acid were enriched in PMA Clostridium strains, which facilitated their adaptation to the unique brewing environment. In addition, horizontal gene transfer contributed to the adaptation of Clostridium to pit mud. Our findings provide genetic insights on PMA Clostridium strains' ecological adaptation and metabolic characteristics.

High-temperature Daqu, usually used as a fermentation starter for sauce-flavor Baijiu production, plays an essential role in the yield and flavor quality of Baijiu. The environmental heterogeneity of different locations in the workshop during fermentation led to the final production of Daqu with three different types (i.e., white, yellow, and black Daqu). How to use these three types of Daqu in Baijiu production mainly depends on the workers' experience so far. Here, we aimed to reveal the potential functions of different types of Daqu by comparing enzyme activity, volatile metabolites, and microbiota characteristics. White_Qu exhibited the highest liquefaction and saccharification enzyme activities, while the highest neutral proteinase and cellulase enzyme activities were detected in black_Qu. The total volatile content of yellow_Qu and black_Qu was roughly double that of white_Qu, and multivariate analysis revealed distinct volatile dissimilarities across different types of Daqu. Significant differences in bacterial and fungal community structures, assembly patterns, and potential functional profiles were discovered among different types of Daqu. At the genus level, Oceanobacillus and Thermomyces dominated the white_Qu microbiota, and the abundant microbes in yellow_Qu and black_Qu were scattered in Kroppenstedtia and Thermoascus. Bacterial and fungal communities were dominated by deterministic and stochastic assembly processes, respectively, suggesting that bacteria may be more affected by abiotic environmental factors and species interaction than fungi. Co-occurrence network analysis showed positive correlations characterized Daqu microbial networks, and network topological features indicated stronger interactions between bacterial taxa compared with fungal community. The Spearman correlation analysis revealed that four bacterial genera (Kroppenstedtia, Virgibacillus, Scopulibacillus, and Staphylococcus) and two fungal genera (Thermoascus and Aspergillus) exhibited positive correlations with almost all of the abundant volatiles. This work reveals that spatially varying environments lead to the microbiome and metabolome heterogeneity of high-temperature Daqu in the same workshop., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mature vinegar culture has usually been used as a type of autochthonous starter to rapidly initiate the next batch of acetic acid fermentation (AAF) and maintain the batch-to-batch uniformity of AAF in the production of traditional cereal vinegar. However, the vitality and dominance of functional microbes in autochthonous starters remain unclear, which hinders further improvement of fermentation yield and production. Here, based on metagenomic (MG), metatranscriptomic (MT), and 16S rRNA gene sequencings, 11 bacterial operational taxonomic units (OTUs) with significant metabolic activity (MT/MG ratio >1) and dominance (relative abundance >1%) were targeted in the autochthonous vinegar starter, all of which were assigned to 4 species (Acetobacter pasteurianus, Lactobacillus acetotolerans, L. helveticus, Acetilactobacillus jinshanensis). Then, we evaluated the successions and interactions of these 11 bacterial OTUs at different AAF stages. Last, a defined starter was constructed with 4 core species isolated from the autochthonous starter (A. pasteurianus, L. acetotolerans, L. helveticus, Ac. jinshanensis). The defined starter culture could rapidly initiate the AAF in a sterile or unsterilized environment, and similar dynamics of metabolites (ethanol, titratable acidity, acetic acid, lactic acid, and volatile compounds) and environmental indexes (temperature, pH) of fermentation were observed as compared with that of autochthonous starter ( P >  0.05). This work provides a method to construct a defined microbiota from a complex system while preserving its metabolic function. IMPORTANCE Complex microorganisms are beneficial to the flavor formation in natural food fermentation, but they also pose challenges to the mass production of standardized products. It is attractive to construct a defined starter to rapidly initiate fermentation process and significantly improve fermentation yield. This study provides a comprehensive understanding of vital and dominant species in the autochthonous vinegar starter via multi-omics, and designs a defined microbial community for the efficient fermentation of cereal vinegar.

Microbial ecosystems of fermented foods are largely interfered by human activities in myriad ways. The aim of this study was to illuminate the impacts of various starters and environmental variables on the fermentation process of Zhenjiang aromatic vinegar (ZAV), one of the four representative cereal vinegars in China. The effects of environmental variables (e.g., ethanol, total acidity, temperature) and starters (e.g., jiuqu, maiqu, seed pei) on the profiles of microbiome and metabolome (e.g., organic acids, amino acids and volatiles) during fermentation process of ZAV were analyzed. Amongst the four fermentation stages, acetic acid fermentation was the main stage for the accumulation of flavor substances, and subsequently, the contents of acids (mainly acetic, lactic and citric acids) and volatile metabolites (e.g., 2,3-butanedione, acetoin, etc.) continued to enrich in sealed fermentation stage. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) showed that the fungal and bacterial community structures of four fermentation stages were significantly different. As for bacterial community, the dominant OTUs with average relative abundance over 10% in at least one fermentation stage were assigned to the genera Acetilactobacillus, Acetobacter, Acinetobacter, Aeromonas, Lactobacillus, and Pseudomonas. The dominant fungal populations in each fermentation stage were obviously divergent, including Wickerhamomyces, Saccharomyces, Alternaria, Fusarium, etc. SourceTracker analysis demonstrated that jiuqu and seed pei provided microorganisms to initiate starch saccharification and acetic acid fermentation stages, respectively, and maiqu was mainly the donor of enzymes in alcohol fermentation. Spearman correlation coefficients revealed positive relationships between fungal community and various flavor metabolites, indicating the essential role of fungi in the flavor formation of ZAV. This study systematically reveals the effects of fermentation starters and environmental variables on vinegar production and deepens the understanding of the traditional production craft., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Chromosomal integration of exogenous genes is preferred for industrially related fermentation, as plasmid-mediated fermentation leads to extra metabolic burden and genetic instability. Moreover, with the development and advancement of genome engineering and gene editing technologies, inserting genes into chromosomes has become more convenient; integration expression is extensively utilized in microorganisms for industrial bioproduction and expected to become the trend of recombinant protein expression. However, in actual research and application, it is important to enhance the expression of heterologous genes at the host genome level. Herein, we summarized the basic principles and characteristics of genomic integration; furthermore, we highlighted strategies to improve the expression of chromosomal integration of genes and pathways in host strains from three aspects, including chassis cell optimization, regulation of expression elements in gene expression cassettes, optimization of gene dose level and integration sites on chromosomes. Moreover, we reviewed and summarized the relevant studies on the application of integrated expression in the exploration of gene function and the various types of industrial microorganism production. Consequently, this review would serve as a reference for the better application of integrated expression.

Daqu, a brick-shaped product spontaneously fermented under an open environment, has been regarded as the starter of fermentation, raw enzyme preparation and raw materials for baijiu production. However, its contribution in baijiu fermentation has not been fully elaborated yet. Here, the effects of daqu microbiota on baijiu fermentation were investigated under both field-scale and lab-scale conditions. In field-scale baijiu fermentation, the dominant daqu microbes (average relative abundance>10.0%), including unclassified&#95;Leuconostocaceae, Thermoascus, and Thermomyces, tended to dominate the early stage (0-7 d). However, the rare daqu microbes (average relative abundance <0.1%, e.g., Kazachstania) tended to dominate the middle and late stages (11-40 d). In addition, some genera showed differences in species diversity between daqu and fermented grains. The average relative abundance of Lactobacillus was over 75% during baijiu fermentation, and most of them were affiliated with Lactobacillus acetotolerans, while Lactobacillus crustorum dominated the Lactobacillus OTUs in daqu. The similar patterns were also observed during lab-scale baijiu fermentation. The results of function prediction showed the enriched metabolic pathways were associated with glycolysis and long-chain fatty acid esters in baijiu fermentation. These results improved the understanding of daqu microbiota function during baijiu fermentation and provided a basic theory to support the regulation of baijiu production., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mud cellars have long been used as anaerobic bioreactors for the fermentation of Chinese strong-flavor Baijiu, where starchy raw materials (mainly sorghum) are metabolized to ethanol and various flavor compounds by multi-species microorganisms. Jiupei (fermented grains) and pit mud are two spatially linked microbial habitats in the mud cellar, yet their metabolic division of labor remains unclear. Here, we investigated the changes in environmental variables (e.g., temperature, oxygen, pH), key metabolites (e.g., ethanol, organic acids) and microbial communities in jiupei and pit mud during fermentation. Jiupei (low pH, high ethanol) and pit mud (neutral pH) provided two habitats with distinctly different environmental conditions for microbial growth. Lactic acid accumulated in jiupei, while butyric and hexanoic acids were mainly produced by microbes inhabiting the pit mud. Biomass analysis using quantitative real-time PCR showed that bacteria dominated the microbial consortia during fermentation, moreover cluster and principal coordinate analysis (PCoA) analysis showed that the bacterial communities of jiupei and pit mud were significantly divergent. The bacterial community diversity of jiupei decreased significantly during the fermentation process, and was relatively stable in pit mud. Lactobacillus dominated the jiupei bacterial community, and its relative abundance reached 98.0% at the end of fermentation. Clostridia (relative abundance: 42.9-85.5%) was the most abundant bacteria in pit mud, mainly distributed in the genus Hydrogenispora (5.3-68.4%). Fungal communities of jiupei and pit mud showed a similar succession pattern, and Kazachstania, Aspergillus and Thermoascus were the predominant genera. PICRUSt analysis demonstrated that enzymes participating in the biosynthesis of acetic and lactic acid were mainly enriched in jiupei samples, while the bacterial community in the pit mud displayed greater potential for butyric and hexanoic acid synthesis. Assays from an in vitro simulated fermentation further validated the roles of jiupei microbiota in acetic and lactic acid production, and these acids were subsequently metabolized to butyric and hexanoic acid by the pit mud microbiota. This work has demonstrated the synergistic cooperation between the microbial communities of jiupei and pit mud for the representative flavor formation of strong-flavor Baijiu., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The mud cellar creates a unique microenvironment for the fermentation of strong-flavor baijiu (SFB). Recent research and long-term practice have highlighted the key roles of microbes inhabiting pit mud in the formation of SFB's characteristic flavor. A positive correlation between the quality of SFB and cellar age was extracted from practice; however, the evolutionary patterns of pit mud microbiome and driving factors remain unclear. Here, based on the variation regularity analysis of microbial community structure and metabolites of samples from cellars of different ages (∼30/100/300 years), we further investigated the effects of lactate and acetate (main microbial metabolites in fermented grains) on modulating the pit mud microbiome. Esters (50.3% to 64.5%) dominated the volatile compounds identified in pit mud, and contents of the four typical acids (lactate, hexanoate, acetate, and butyrate) increased with cellar age. Bacteria (9.5 to 10.4 log 10 [lg] copies/g) and archaea (8.3 to 9.1 lg copies/g) mainly constituted pit mud microbiota, respectively dominated by Clostridia (39.7% to 81.2%) and Methanomicrobia (32.8% to 92.9%). An upward trend with cellar age characterized the relative and absolute abundance of the most predominant bacterial and archaeal genera, Caproiciproducens and Methanosarcina . Correlation analysis revealed significantly ( P  < 0.05) positive relationships between the two genera and major metabolites. Anaerobic fermentation with acetate and lactate as carbon sources enhanced the enrichment of Clostridia , and furthermore, the relative abundance of Caproiciproducens (40.9%) significantly increased after 15-day fed-batch fermentation with lactate compared with the initial pit mud (0.22%). This work presents a directional evolutionary pattern of pit mud microbial consortia and provides an alternative way to accelerate the enrichment of functional microbes. IMPORTANCE The solid-state anaerobic fermentation in a mud cellar is the most typical feature of strong-flavor baijiu (SFB). Metabolites produced by microbes inhabiting pit mud are crucial to create the unique flavor of SFB. Accordingly, craftspeople have always highlighted the importance of the pit mud microbiome and concluded by centuries of practice that the production rate of high-quality baijiu increases with cellar age. To deepen the understanding of the pit mud microbiome, we determined the microbial community and metabolites of different-aged pit mud, inferred the main functional groups, and explored the forces driving the microbial community evolution through metagenomic, metabolomic, and multivariate statistical analyses. The results showed that the microbial consortia of pit mud presented a regular and directional evolutionary pattern under the impact of continuous batch-to-batch brewing activities. This work provides insight into the key roles of the pit mud microbiome in SFB production and supports the production optimization of high-quality pit mud.

As a widely used Asian starter culture, the quality of daqu can significantly affect the organoleptic characteristics of the final products, yet the microbial metabolic network involved in flavor development remains unclear. This study aims to investigate that network based on the dynamics of physicochemical properties, microbial community, and volatile compounds in medium-temperature daqu (MT-daqu) during spontaneous fermentation. Analyses using the metagenomic data set facilitated the gene repertoire overview of this ecosystem, indicating that Lactobacillales (mainly Weissella, Lactobacillus, and Pediococcus), Mucorales (mainly Lichtheimia), and Eurotiales (mainly Aspergillus, Rasamsonia and Byssochlamys) were the potential predominant populations successively responsible for the production of lytic enzymes and flavor precursors/compounds in MT-daqu. Flavor-relevant pathways were found to exist in multiple species, but only bacteria showed the potential to participate in butane-2,3-diol (e.g. Weissella, Lactobacillus, and Staphylococcus) and butanoate (Thermoactinomyces) metabolism, and only fungi were potentially involved in biosynthesis of guaiacol (Byssochlamys) and 4-vinylguaiacol (Aspergillus). Furthermore, a combined analysis revealed that the acidic thermal environment present in early phases was mainly due to the catabolic activities of Lactobacillales and Lichtheimia, which could contribute to the effective self-domestication of microbiota. The study helps elucidate the different metabolic roles of microorganisms and disclose the formation mechanism of daqu's partial functions, namely providing various aromatic substances/precursors and enzymes., (Copyright © 2020. Published by Elsevier Ltd.)

Acetoin, giving a creamy yogurt aroma and buttery taste, exists in cereal vinegar as an important flavor substance and is mainly produced by the metabolism of Lactobacillus and Acetobacter during multispecies solid-state acetic acid fermentation. However, the impacts of Lactobacillus-Acetobacter interactions on acetoin accumulation and the microbial metabolism during acetic acid fermentation are not completely clear. Here, six strains isolated from vinegar fermentation culture and associated with acetoin metabolism, namely, Lactobacillus reuteri L-0, L. buchneri F2-6, L. brevis 4-20, L. fermentum M10-7, L. casei M1-6 and Acetobacter pasteurianus G3-2, were selected for microbial growth and metabolism analysis in monoculture and coculture fermentations. Lactobacillus sp. and A. pasteurianus G3-2 respectively utilized glucose and ethanol preferentially. In monocultures, L. casei M1-6 (183.7 mg/L) and A. pasteurianus G3-2 (121.0 mg/L) showed better acetoin-producing capacity than the others. In the bicultures with Lactobacillus sp. and A. pasteurianus G3-2, biomass analysis in the stationary phase demonstrated that significant growth depressions of Lactobacillus sp. occurred compared with monocultures, possibly due to intolerance to acetic acid produced by A. pasteurianus G3-2. Synergistic effect between Lactobacillus sp. and A. pasteurianus G3-2 on enhanced acetoin accumulation was identified, however, cocultures of two Lactobacillus strains could not apparently facilitate acetoin accumulation. Coculture of L. casei M1-6 and A. pasteurianus G3-2 showed the best performance in acetoin production amongst all mono-, bi- and triculture combinations, and the yield of acetoin increased from 1827.7 to 7529.8 mg/L following optimization of culture conditions. Moreover, the interactions of L. casei M1-6 and A. pasteurianus G3-2 regulated the global metabolism of vinegar microbiota during fermentation through performing in situ bioaugmentation, which could accelerate the production of acetic acid, lactic acid, acetoin, ethyl acetate, ethyl lactate, ligustrazine and other important flavoring substances. This work provides a promising strategy for the production of acetoin-rich vinegar through Lactobacillus sp.-A. pasteurianus joint bioaugmentation., (Copyright © 2020. Published by Elsevier Ltd.)

Symphony orchestra of multi-microorganisms characterizes the solid-state acetic acid fermentation process of Chinese cereal vinegars. Lactate is the predominant non-volatile acid and plays indispensable roles in flavor formation. This study investigated the microbial consortia driving the metabolism of D-/l-lactate during fermentation. Sequencing analysis based on D-/l-lactate dehydrogenase genes demonstrated that Lactobacillus (relative abundance: > 95%) dominated the production of both d-lactate and l-lactate, showing species-specific features between the two types. Lactobacillus helveticus (>65%) and L. reuteri (~80%) respectively dominated l- and d-lactate-producing communities. D-/l-lactate production and utilization capabilities of eight predominant Lactobacillus strains were determined by culture-dependent approach. Subsequently, D-/l-lactate producer L. plantarum M10-1 (d:l ≈ 1:1), l-lactate producer L. casei 21M3-1 (D:L ≈ 0.2:9.8) and D-/l-lactate utilizer Acetobacter pasteurianus G3-2 were selected to modulate the metabolic flux of D-/l-lactate of microbial consortia. The production ratio of D-/l-lactate was correspondingly shifted coupling with microbial consortia changes. Bioaugmentation with L.casei 21M3-1 merely enhanced l-lactate production, displaying ~4-fold elevation at the end of fermentation. Addition of L.plantarum M10-1 twice increased both D- and l-lactate production, while A. pasteurianus G3-2 decreased the content of D-/l-isomer. Our results provided an alternative strategy to specifically manipulate the metabolic flux within microbial consortia of certain ecological niches., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people's health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera ( Staphylococcus , Bacillus , Weissella , Aspergillus , and Zygosaccharomyces ) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains ( Aspergillus oryzae , Bacillus subtilis , Staphylococcus gallinarum , Weissella confusa , and Zygosaccharomyces rouxii ) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function. IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment., (Copyright © 2020 American Society for Microbiology.)

Over thousands of years, humans have mastered the natural vinegar fermentation technique of cultivating functional microbiota on different raw materials. Functional microbial communities that form reproducibly on non-autoclaved raw materials through repeated batch acetic acid fermentation underpin the flavour development of traditional cereal vinegars. However, how to modulate rationally and optimise the metabolic function of these naturally engineered acidic ecosystems remains unclear. Exploring two key minorities in a vinegar ecosystem, including microbial functions (e.g., flavour and aroma synthesis) and microbial strains, is a crucial step for the vinegar industry to modulate the metabolic function of vinegar microbiota, to monitor the fermentation process, and to maintain the flavour quality of final product., (Copyright © 2017. Published by Elsevier Ltd.)

"Daqu" is a saccharifying and fermenting agent commonly used in the traditional solid-state fermentation industry (e.g., baijiu and vinegar). The patterns of microbial community succession and flavor formation are highly similar among batches, yet the mechanisms promoting temporal succession in the Daqu microbial ecology remain unclear. Here, we first correlated temporal profiles of microbial community succession with environmental variables (temperature, moisture, and titratable acidity) in medium temperature Daqu (MT-Daqu) throughout fermentation. Temperature dynamics significantly correlated ( P < 0.05) with the quick succession of MT-Daqu microbiota in the first 12 d of fermentation, while the community structure was relatively stable after 12 d. Then, we explored the effect of temperature on the MT-Daqu community assembly. In the first 4 d of fermentation, the rapid propagation of most bacterial taxa and several fungal taxa, including Candida , Wickerhamomyces , and unclassified Dipodascaceae and Saccharomycetales species, significantly increased MT-Daqu temperature to 55°C. Subsequently, sustained bio-heat generated by microbial metabolism (53 to 56°C) within MT-Daqu inhibited the growth of most microbes from day 4 to day 12, while thermotolerant taxa, including Bacillus , unclassified Streptophyta , Weissella , Thermoactinomyces , Thermoascus , and Thermomyces survived or kept on growing. Furthermore, temperature as a major driving force on the shaping of MT-Daqu microbiota was validated. Lowering the fermentation temperature by placing the MT-Daqu in a 37°C incubator resulted in decreased relative abundances of thermotolerant taxa, including Bacillus , Thermoactinomyces , and Thermoascus , in the MT-Daqu microbiota. This study revealed that bio-heat functioned as a primary endogenous driver promoting the formation of functional MT-Daqu microbiota. IMPORTANCE Humans have mastered the Daqu preparation technique of cultivating functional microbiota on starchy grains over thousands of years, and it is well known that the metabolic activity of these microbes is key to the flavor production of Chinese baijiu. The pattern of microbial community succession and flavor formation remains highly similar between batches, yet mechanistic insight into these patterns and into microbial population fidelity to specific environmental conditions remains unclear. Our study revealed that bio-heat was generated within Daqu bricks in the first 4 d of fermentation, concomitant with rapid microbial propagation and metabolism. The sustained bio-heat may then function as a major endogenous driving force promoting the formation of the MT-Daqu microbiota from day 4 to day 12. The bio-heat-driven growth of thermotolerant microorganisms might contribute to the formation of flavor metabolites. This study provides useful information for the temperature-based modulation of microbiota function during the fermentation of Daqu., (Copyright © 2017 American Society for Microbiology.)

Multispecies microbial community formed through centuries of repeated batch acetic acid fermentation (AAF) is crucial for the flavour quality of traditional vinegar produced from cereals. However, the metabolism to generate and/or formulate the essential flavours by the multispecies microbial community is hardly understood. Here we used metagenomic approach to clarify in situ metabolic network of key microbes responsible for flavour synthesis of a typical cereal vinegar, Zhenjiang aromatic vinegar, produced by solid-state fermentation. First, we identified 3 organic acids, 7 amino acids, and 20 volatiles as dominant vinegar metabolites. Second, we revealed taxonomic and functional composition of the microbiota by metagenomic shotgun sequencing. A total of 86 201 predicted protein-coding genes from 35 phyla (951 genera) were involved in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of Metabolism (42.3%), Genetic Information Processing (28.3%), and Environmental Information Processing (10.1%). Furthermore, a metabolic network for substrate breakdown and dominant flavour formation in vinegar microbiota was constructed, and microbial distribution discrepancy in different metabolic pathways was charted. This study helps elucidating different metabolic roles of microbes during flavour formation in vinegar microbiota., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.