The initial composition and structure of microbial community determined the yield and quality of Baijiu during the spontaneous fermentation / La composición inicial y la estructura de la comunidad microbiana determinaron el rendimiento y la calidad del Baijiu durante la fermentación espontánea

The microbiota during pit mud fermentation is a crucial factor in Baijiu brewing since it determines the yield and flavor. However, the impact of the microbial community during the initial fermentation stage on Baijiu quality remains uncertain. Herein, high-throughput sequencing was employed to investigate the microbial diversities and distribution during Baijiu fermentation in individual pit mud workshops at both initial and late stages. During the initial fermentation stage, the bacterial community exerted a more pronounced effect on Baijiu quality than the fungal community. And the high-yield pit mud workshop exhibited lower richness and evenness, as well as greater Bray-Curtis dissimilarity during Baijiu fermentation. Lactobacillus was the dominant genus and biomarker in high-yield pit mud, and it constituted the only genus within the bacterial association network during the late fermentation stage. Fungal communities tended to maintain a simple association network with selected core species. Based on the correlation network, Rhizopus and Trichosporon were identified as biomarkers in Baijiu fermentation process. Together, Lactobacillus and Rhizopus could serve as bio-indicators for Baijiu quality during the initial fermentation stage. Therefore, these findings provided novel insights into microbiota interactions during fermentation and the impact of initial microbiota on final Baijiu quality.(AU)

Microbiologic surveys for Baijiu fermentation are affected by experimental design.

The traditional Chinese alcoholic beverage Baijiu is produced by spontaneous fermentation of grains under anaerobic conditions. While numerous studies have used metagenomic technology to investigate the microbiome of Baijiu brewing, the microbial succession mechanism of Baijiu brewing has not been fully clarified, and metagenomic strategies for microecology surveys have not been comprehensively evaluated. Using the fermentation process of strong-flavor Baijiu as a model, we compared the data for bacterial communities based on short read 16S rRNA variable regions, V3-V4, and full-length 16S regions, V1-V9, to whole metagenomic shotgun sequencing (WMS) to measure the effect of technology selection on phylogenetic and functional profiles. The results showed differences in bacterial compositions and their relation to volatiles and physicochemical variables between sequencing methods. Furthermore, the percentage of V3-V4 sequences assigned to species level was higher than the percentage of V1-V9 sequences according to SILVA taxonomy, but lower according to NCBI taxonomy (P < 0.05). In both SILVA and NCBI taxonomies, the relative abundances of bacterial communities at both the genus and family levels were more positively correlated with WMS data in the V3-V4 dataset than in the V1-V9 dataset. The WMS identified changes in abundances of multiple metabolic pathways during fermentation (P < 0.05), including "starch and sucrose metabolism," "galactose metabolism," and "fatty acid biosynthesis." Although functional predictions derived from 16S data show similar patterns to WMS, most metabolic pathway changes are uncorrelated (P > 0.05). This study provided new technical and biological insights into Baijiu production that may assist in selection of methodologies for studies of fermentation systems.

Exploring the heterogeneity of community and function and correspondence of "species-enzymes" among three types of Daqu with different fermentation peak-temperature via high-throughput sequencing and metagenomics.

The enzyme activity of Daqu is an important prerequisite for defining it as a Baijiu starter. However, little is known about the functional species related to enzymes in different types of Daqu at the metagenomic level. Therefore, we analyzed the differences in enzymatic properties, microbial composition and metabolic function of three types of Daqu, namely high-, medium- and low-temperature Daqus (HTD, MTD and LTD), by combining chemical feature and multi-dimensional sequencing. The results showed that both liquefaction, saccharification, fermentation and esterification powers were remarkably weaker in HTD compared to MTD and LTD. Totally, 30 bacterial and 5 fungal phyla were identified and significant differences in community structures were also observed among samples, with Brevibacterium/Microascus, Pseudomonas, and Lactobacillus/Saccharomycopsis identified as biomarkers for HTD, MTD and LTD, respectively. Additionally, the importance of deterministic assembly in bacterial communities was proportional to the fermentation peak-temperature, while stochastic assembly dominated in fungal ones. Metagenomics analysis indicated eukaryota (>80 %, mainly Ascomycota) predominated in HTD and MTD while bacteria (54.3 %, mainly Actinobacteriota) were more abundant in LTD. However, the functional profiles and pathways of MTD and LTD were more similar, and the synthesis and metabolism of carbohydrates and amino acids were the crucial biological functions of all samples. Finally, the relationship between species and enzymes in different samples was constructed and the functional species in LTD and MTD were more diverse than HTD, which elucidated the functional species associated with enzyme activity in each type of Daqu. These results will greatly enrich our understanding of the core functional species in three typical Daqu, which provide available information for rational regulation of Daqu quality and the Baijiu fermentation.

The initial composition and structure of microbial community determined the yield and quality of Baijiu during the spontaneous fermentation.

The microbiota during pit mud fermentation is a crucial factor in Baijiu brewing since it determines the yield and flavor. However, the impact of the microbial community during the initial fermentation stage on Baijiu quality remains uncertain. Herein, high-throughput sequencing was employed to investigate the microbial diversities and distribution during Baijiu fermentation in individual pit mud workshops at both initial and late stages. During the initial fermentation stage, the bacterial community exerted a more pronounced effect on Baijiu quality than the fungal community. And the high-yield pit mud workshop exhibited lower richness and evenness, as well as greater Bray-Curtis dissimilarity during Baijiu fermentation. Lactobacillus was the dominant genus and biomarker in high-yield pit mud, and it constituted the only genus within the bacterial association network during the late fermentation stage. Fungal communities tended to maintain a simple association network with selected core species. Based on the correlation network, Rhizopus and Trichosporon were identified as biomarkers in Baijiu fermentation process. Together, Lactobacillus and Rhizopus could serve as bio-indicators for Baijiu quality during the initial fermentation stage. Therefore, these findings provided novel insights into microbiota interactions during fermentation and the impact of initial microbiota on final Baijiu quality.

Yeast diversity in open agave fermentations across Mexico.

Yeasts are a diverse group of fungal microorganisms that are widely used to produce fermented foods and beverages. In Mexico, open fermentations are used to obtain spirits from agave plants. Despite the prevalence of this traditional practice throughout the country, yeasts have only been isolated and studied from a limited number of distilleries. To systematically describe the diversity of yeast species from open agave fermentations, here we generate the YMX-1.0 culture collection by isolating 4524 strains from 68 sites with diverse climatic, geographical, and biological contexts. We used MALDI-TOF mass spectrometry for taxonomic classification and validated a subset of the strains by ITS and D1/D2 sequencing, which also revealed two potential novel species of Saccharomycetales. Overall, the composition of yeast communities was weakly associated with local variables and types of climate, yet a core set of six species was consistently isolated from most producing regions. To explore the intraspecific variation of the yeasts from agave fermentations, we sequenced the genomes of four isolates of the nonconventional yeast Kazachstania humilis. The genomes of these four strains were substantially distinct from a European isolate of the same species, suggesting that they may belong to different populations. Our work contributes to the understanding and conservation of an open fermentation system of great cultural and economic importance, providing a valuable resource to study the biology and genetic diversity of microorganisms living at the interface of natural and human-associated environments.

Qu-omics elucidates the formation and spatio-temporal differentiation mechanism underlying the microecology of high temperature Daqu.

Even if fermented in the same qu-room, Daqu will form various microecologies. A gradual differentiation of microbial population niches was observed within different qu-layers, manifesting as variations in the abundance of dominant microorganisms including Bacillus, Saccharopolyspora, Weissella, Kroppenstedtia, Thermoascus, Thermomyces, Saccharomycopsis, and Rasamsonia. Moreover, distinctions were observed in the functional expression of microorganisms, including Aspergillus, Virgibacillus, Oceanobacillus, and Neurospora. The community in middle layer Daqu exhibited characteristics of high compactness and niche diversity, which facilitated efficient substrate utilization. During the initial phase, the upper Daqu community demonstrated heightened activity. However, in the middle and lower layers, fungi and bacteria respectively exhibited greater functional expression. The key environmental factors regulating the assembly of communities in the upper and middle layers were pH and temperature, respectively, and the lower was moisture and acidity. Notably, deterministic assembly exerted a stronger influence on fungi.

Effect of Caproicibacterium lactatifermentans inoculation on the microbial succession and flavor formation of pit mud used in Chinese Baijiu fermentation.

Caproicibacterium lactatifermentans is a major caproate-producing bacterium in high-quality pit mud and has an impact on the synthesis of fatty acids during Baijiu fermentation. To develop an effective method for cultivating high-quality pit mud, we explored the role of Caproicibacterium lactatifermentans inoculation. The inoculation resulted in a high level of Caproicibacterium lactatifermentans (29.16%) and fortified pit mud produced abundant fatty acids and ethyl esters in short-term usage. Rare microbes, such as Hazenella coriacea, promoted the production of fatty acids. After long-term usage, changes in physicochemical properties led to a decrease in caproate-producing bacterium, namely Clostridium and Caproicibacterium, and an increase in microbes with limited fatty acid biosynthesis capability, including Proteiniphilum, Fastidiosipila, and Caldicoprobacter. These alterations ultimately led to a decrease in fatty acids and ethyl esters. In summary, Caproicibacterium lactatifermentans inoculation exhibited positive outcomes in obtaining high-quality pit mud. However, the maintenance of functional microbes necessitates further investigation.

Ultra-high-depth macrogenomic sequencing revealed differences in microbial composition and function between high temperature and medium-high temperature Daqu.

Complex microorganisms in Daqu of different temperatures play a vital role in the taste, flavor and quality of Baijiu during fermentation. However, understanding the functional diversity of the whole microbial community between the Daqus of two different temperatures (high temperature Daqu, HD and medium-high temperature Daqu, MD) remains a major challenge. Here, a systematic study of the microbial diversity, functions as well as physiological and biochemical indexes of Daqu are described. The results revealed that the Daqu exhibited unique characteristics. In particular, the diversity of microorganisms in HD and MD was high, with 44 species including 14 novel species (Sphingomonas sp. is the main novel species) detected in all samples. Their profiles of carbohydrate-active enzymes and specific functional components supported the fact that these species were involved in flavor formation. The Daqu microbiome consisted of a high proportion of phage, providing evidence of phage infection/genome integration and horizontal gene transfer from phage to bacteria. Such processes would also regulate Daqu microbiomes and thus flavor quality. These results enrich current knowledge of Daqu and can be used to promote the development of Baijiu fermentation technology.

Effect of inoculated Daqu on the spontaneous fermentation of Chinese liquor.

Inoculated fermentation is widely used to improve the efficiency and quality of food production. However, it is still unclear how the inoculated multi-species starters influence food fermentation. Here we prepared two different types of Daqu, with/without the inoculation of Bacillus licheniformis in spontaneous Daqu fermentation, and studied their effects on liquor fermentation. These two types of Daqu were different in microbial community, and the inoculated Daqu had significantly higher relative abundance of Bacillus (69.2%) and lower relative abundance of Lactobacillus (3.2%) than those of Daqu without inoculation (Bacillus, 13.5%; Lactobacillus, 14.0%). After using with these two types of Daqu, metatranscriptomic analysis revealed that Kazachstania, Naumovozyma, Saccharomyces, Nakaseomyces and Lactobacillus were the transcriptional active genera during liquor fermentation. The transcription of Lactobacillus decreased on days 10 and 20 in liquor fermentation with the inoculated Daqu. The transcription of Kazachstania, Naumovozyma and Saccharomyces decreased on day 10 but increased on day 20 with the inoculated Daqu. Although lactate dehydrogenase decreased in Lactobacillus, alcohol dehydrogenase, aldehyde dehydrogenase and lactate dehydrogenase increased in Saccharomyces on day 20 in fermentation with inoculated Daqu, it indicated an extended succession of Saccharomyces in liquor fermentation. This would facilitate the increase of ethanol, acetic acid and lactic acid contents in liquor fermentation with inoculated Daqu. This work would be beneficial for improving Chinese liquor fermentation.

Lentibacillus daqui sp. nov., isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS110521T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu and was characterised by polyphasic taxonomy. This novel isolate grew in the presence of 0-20 % (w/v) NaCl, at pH 6.0-9.0 and 20-50 °C; optimum growth was observed with 8-10 % (w/v) NaCl, at pH 7.0 and 37 °C. A comparative analysis of the 16S rRNA gene sequence (1460 bp) of ZS110521T revealed that it displayed the highest similarity to Lentibacillus populi WD4L-1T (95.5 %), followed by Lentibacillus garicola SL-MJ1T (95.4 %) and Lentibacillus lacisalsi BH260T (95.2 %). ANI and dDDH values between ZS110521T and other strains of species of the genus Lentibacillus were less than 78 and 28 %, respectively. The predominant cellular fatty acids (> 10 %) of ZS110521T were anteiso-C17 : 0 (37.8 %), anteiso-C15 : 0 (28.1 %) and iso-C16 : 0 (15.5 %). The respiratory quinone was identified as menaquinone-7 (MK-7) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The polyphasic taxonomic data and the results of chemotaxonomic analysis confirmed that ZS110521T represents a novel species, for which the name Lentibacillus daqui sp. nov. is proposed. The type strain of this proposed species is ZS110521T (=CGMCC 1.19456T =JCM 35213T).

Pontibacter liquoris sp. nov. and Pontibacter vulgaris sp. nov., two novel bacteria isolated from the pit mud of Chinese liquor.

Two pink-pigmented bacterial strains, designated NBU2971T and NBU2972T, were isolated from the pit mud of a Chinese liquor. Phylogenetic analyses based on 16S rRNA gene sequences suggested that strains NBU2971T and NBU2972T formed a distinct lineage within the family Hymenobacteraceae and were closely related to members of the genus Pontibacter. 16S rRNA gene sequences revealed that strain NBU2971T showed highest similarity of 97.9 % to Pontibacter arcticus 2b14T, and strain NBU2972T showed the highest similarity of 96.9 % to Pontibacter deserti JC215T. The 16S rRNA gene sequence similarity, average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the two novel strains were 95.2, 73.8 and 19.6 %, respectively, suggesting that they represent different species. The ANI and dDDH values between two novel strains and related species of genus Pontibacter were well below the threshold limit for prokaryotic species delineation. The genomic DNA G+C contents of strains NBU2971T and NBU2972T were 51.3 and 44.5 mol%, respectively. The major cellular fatty acids of the two novel strains were iso-C15 : 0 and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The major polar lipid of both novel strains was phosphatidylethanolamine. The only respiratory quinone was MK-7. Combining results of phenotypic, chemotaxonomic and genotypic data, strains NBU2971T and NBU2972T are considered to be two representatives in the genus Pontibacter, which the name Pontibacter liquoris sp. nov. and Pontibacter vulgaris sp. nov. are proposed. The type strains of the new species are NBU2971T (=KCTC 82916T=MCCC 1K06395T) and NBU2972T (=KCTC 82917T=MCCC 1K06396T), respectively.

Effect of lactic acid bacteria on the structure and potential function of the microbial community of Nongxiangxing Daqu.

OBJECTIVES: The microbial community structure of the saccharifying starter, Nongxiangxing Daqu(Daqu), is a crucial factor in determining Baijiu's quality. Lactic acid bacteria (LAB), are the dominant microorganisms in the Daqu. The present study investigated the effects of LAB on the microbial community structure and its contribution to microbial community function during the fermentation of Daqu. METHODS: The effect of LAB on the structure and function of the microbial community of Daqu was investigated using high-throughput sequencing technology combined with multivariate statistical analysis. RESULTS: LAB showed a significant stage-specific evolution pattern during Daqu fermentation. The LEfSe analysis and the random forest learning algorithm identified LAB as vital differential microorganisms during Daqu fermentation. The correlation co-occurrence network showed aggregation of LAB and Daqu microorganisms, indicating LAB's significant position in influencing the microbial community structure, and suggests that LAB showed negative correlations with Bacillus, Saccharopolyspora, and Thermoactinomyces but positive correlations with Issatchenkia, Candida, Acetobacter, and Gluconobacter. The predicted genes of LAB enriched 20 functional pathways during Daqu fermentation, including Biosynthesis of amino acids, Alanine, aspartate and glutamate metabolism, Valine, leucine and isoleucine biosynthesis and Starch and sucrose metabolism, which suggested that LAB had the functions of polysaccharide metabolism and amino acid biosynthesis. CONCLUSION: LAB are important in determining the composition and function of Daqu microorganisms, and LAB are closely related to the production of nitrogenous flavor substances in Daqu. The study provides a foundation for further exploring the function of LAB and the regulation of Daqu quality.

Effect of kuratsuki Kocuria on sake's taste varies depending on the sake yeast strain used in sake brewing.

Although sake yeast mainly produces the taste of sake, sake brewery-inhabiting (kuratsuki) bacteria affect the taste of sake. Thus, kuratsuki bacteria may alter the metabolism of sake yeast through interactions between kuratsuki bacteria and sake yeast. This study aimed to confirm the effects of the combination of kuratsuki Kocuria TGY1127_2 and different sake yeast strains, AK25, K901, and K1801 on the taste of sake. Although the Brix and acidity during sake production using AK25 differed between sake with and without kuratsuki Kocuria, those using K901 and K1801 did not differ. Thus, sake yeast AK25 interacted with kuratsuki Kocuria and changed its characteristics of ethanol fermentation. In addition, the taste intensity changes, measured with a taste sensor TS-5000Z, showed that the effects of adding kuratsuki Kocuria varied among different sake yeasts. Thus, each sake yeast strain interacted with the kuratsuki bacterium and produced different metabolites, resulting in a change in the taste of sake. The findings of this study can lead to the brewing of sake using different types of kuratsuki bacteria which can affect the taste of sake.

Evaluation of microbial communities of Chinese Feng-flavor Daqu with effects of environmental factors using traceability analysis.

Analysis of the changes of microorganisms during Chinese Feng-flavor Daqu fermentation, and the specific contribution of different environmental factors to Daqu microorganisms. High throughput sequencing technology and SourceTracker software were used to analyze the microbial diversity of Feng-flavor Daqu before and after fermentation. 85 fungal and 105 bacterial were detected in the newly pressed Feng-flavor Daqu, while 33 fungal and 50 bacterial in the mature Daqu, and 202 fungal and 555 bacterial in the environmental samples. After fermentation, the microbial community structure of Daqu changed and decreased significantly. 94.7% of fungi come from raw materials and 1.8% from outdoor ground, 60.95% of bacteria come from indoor ground, 20.44% from raw materials, and 8.98% from tools. By comparing the changes of microorganisms in Daqu before and after fermentation, the microorganisms in mature Daqu may mainly come from not only the enhanced strains but also the environment.The source of main microorganisms in Feng-flavor Daqu and the influence of environmental factors on the quality of Daqu were clarified, which provided a basis for improving the quality of Feng-flavor Daqu.

Characterization of the heteropolysaccharides produced by Liquorilactobacillus sicerae CUPV261 and Secundilactobacillus collinoides CUPV237 isolated from cider.

Some lactic acid bacteria (LAB) strains isolated from alcoholic beverages are able to produce exopolysaccharides (EPS). The present work focuses on the physico-chemical characterization of the heteropolysaccharides (HePS) produced by Liquorilactobacillus sicerae CUPV261T (formerly known as Lactobacillus sicerae) and Secundilactobacillus collinoides CUPV237 (formerly known as Lactobacillus collinoides) strains isolated from cider. Genome sequencing and assembly enabled the identification of at least four putative HePS gene clusters in each strain, which correlated with the ability of both strains to secrete EPS. The crude EPS preparation from CUPV261T contained glucose, galactose and rhamnose, and that of CUPV237 was composed of glucose, galactose and N-acetylglucosamine. Both EPS were mixtures of HePS of different composition, with two major soluble components of average molecular weights (Mw) in the range of 106 and 104 g.mol-1. These HePS were resistant to gastric stress conditions in an in vitro model, and they significantly reduced zebrafish larvae mortality in an in vivo model of inflammatory bowel disease.

Variation in microbiological heterogeneity in Chinese strong-flavor Baijiu fermentation for four representative varieties of sorghum.

The quality and composition of strong-flavor Baijiu (SFB), a type of Chinese liquor, depends on the variety of sorghum used in fermentation. However, comprehensive in situ studies measuring the effects sorghum varieties on the fermentation are lacking and the underlying microbial mechanisms remains poorly understood. We studied the in situ fermentation of SFB by using metagenomic, metaproteomic, and metabolomic techniques across four sorghum varieties. Sensory characteristics were best for SFB made from glutinous variety Luzhouhong, followed by glutinous hybrid Jinnuoliang and Jinuoliang, and those made with non-glutinous Dongzajiao. In agreement with sensory evaluations, the volatile composition of SFB samples differed between sorghum varieties (P < 0.05). Fermentation of different sorghum varieties varied in microbial diversity, structure, volatile compounds, and physicochemical properties (pH, temperature, starch, reducing sugar, and moisture) (P < 0.05), with most changes occurring within the first 21 days. Additionally, the microbial interactions and their relationship with volatiles, as well as the physicochemical factors that govern microbial succession, differed between varieties of sorghum. The number of physicochemical factors affecting bacterial communities outweighed those affecting fungal communities, suggesting that bacteria were less resilient to the brewing conditions. This correlates with the finding that bacteria play a major role in the differences in microbial communities and metabolic functions during fermentation with the different varieties of sorghum. Metagenomic function analysis revealed differences in amino acid and carbohydrate metabolism between sorghum varieties throughout most of the brewing process. Metaproteomics further indicated most differential proteins were found in these two pathways, related to differences in volatiles between sorghum varieties of Baijiu and originating from Lactobacillus. These results provide insight into the microbial principles underlying Baijiu production and can be used to improve the quality of Baijiu by selecting the appropriate raw materials and optimizing fermentation parameters.

Effect of kuratsuki Kocuria on sake brewing in different koji conditions.

Koji is made using steamed rice and a koji mold, which plays an essential role in sake brewing. We challenge to build a new sake brewing method using the kuratsuki bacteria that have inhabited each sake brewery. In this paper, effects of the kuratsuki Kocuria strain TGY1127_2 were estimated on sake brewing in different koji conditions. Sake was produced by incubation of a mixture solution of koji, water, and sake yeast (strain K1401) with and without the kuratsuki Kocuria TGY1127_2. The effects of the kuratsuki Kocuria on the taste of the sake differed among different koji. The kuratsuki Kocuria led to an increase in ethanol concentration. Additionally, the sugar content (Brix) and acidity of the sake increased in proportion to the amount of koji. These results strongly suggest that the kuratsuki Kocuria does not adversely affect the fermentation activity of the sake yeast. Thus, the kuratsuki Kocuria had different effects on the taste of sake among different koji but the fermentation activity of the sake yeast was maintained.

Rare short- and medium-chain fatty acid-producing anaerobes from raw soil play vital roles in formation of diverse flavour compounds of Jiangxiangxing Baijiu.

Pit mud is an essential habitat for diverse anaerobes, however, how pit mud of Jiangxiangxing Baijiu contributes to flavour is still unclear. The correlation between pit mud anaerobes and flavour compounds formation was investigated by analyzing flavour compounds and prokaryotic community of pit mud as well as fermented grains. Then scaling-down fermentation and culture-dependent approach were used to verify the effects of pit mud anaerobes on flavour compound formation. We found that short- and medium-chain fatty acids and alcohols, e.g., propionate, butyrate, caproate, 1-butanol, 1-hexanol, and 1-heptanol, were the vital flavour compounds produced by pit mud anaerobes. Pit mud anaerobes hardly migrated into fermented grains because of the low pH and low moisture of fermented grains. Therefore, the flavour compounds produced by pit mud anaerobes might enter fermented grains via volatilization. Moreover, enrichment culturing proved that raw soil was one of the sources for pit mud anaerobes, e.g., Clostridiumtyrobutyricum, Ruminococcaceae bacterium BL-4 and Caproicibacteriumamylolyticum. These rare short- and medium-chain fatty acid-producing anaerobes in raw soil can be enriched during Jiangxiangxing Baijiu fermentation. These findings clarified the role of pit mud during Jiangxiangxing Baijiu fermentation and revealed the key species involved in short- and medium-chain fatty acid-producing production.

Effects of Daqu properties on the microbial community and their metabolites in fermented grains in Baijiu fermentation system.

Daqu is the natural starter for Nong-flavor Baijiu brewing. The effects of Daqu properties on the microbial community succession and their metabolites in fermented grains (FG) during Baijiu brewing were determined. These results showed that the effect of Daqu on the bacterial communities was stronger than that of the fungal communities. Compared with the conventional Daqu (DZ), Taikong (TK), and Qianghua (QH), Daqu significantly enhanced the content of volatile metabolites (especially esters) and ethanol when they were used, respectively, for FG fermentation. In the second round of fermentation, the relative abundance of Lactobacillus decreased, the content of lactic acid decreased, and that of caproic acid increased. In particular, the abundance of Lactobacillus was also reduced by 20% in FGs of the second round when TK Daqu was used than that in the respective first round. Partial least squares structural equation model analysis also showed that physicochemical parameters and Daqu properties significantly affected FG community structure and metabolism. This study provides a theoretical basis for further study on the effect of high-quality Daqu on the quality of fresh Baijiu and lays an important theoretical foundation for the stabilization of the Baijiu fermentation system based on Daqu.

Bacteria and filamentous fungi running a relay race in Daqu fermentation enable macromolecular degradation and flavor substance formation.

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.