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The unique cellar fermentation process of Chinese strong-flavor Baijiu is the reason for its characteristic cellar aroma flavor. The types, abundance, community structure and metabolic activity of microorganisms in the pit mud directly affect the microbial balance in the white spirit production environment, promoting the formation of typical aromas and influencing the quality of CFSB. During the production process, the production of off-flavor in the cellar may occur. The aim of this study is to elucidate the differences in microbiota and flavor between normal pit mud and abnormal pit mud (pit mud with off-flavor). A total of 46 major volatile compounds were identified, and 24 bacterial genera and 21 fungal genera were screened. The esters, acids, and alcohols in the abnormal pit mud were lower than those in the normal pit mud, while the aldehydes were higher. 3-Methyl indole, which has been proven to be responsible for the muddy and musty flavors, was detected in both types of pit mud, and for the first time, high levels of 4-methylanisole was detected in the pit mud. The microbial composition of the two types of pit mud showed significant differences in the bacterial genera of Sporosarcina , Lactobacillus , Garciella , Anaerosalibacter , Lentimicrobium , HN–HF0106, Petrimonas , Clostridium_sensu_stricto_12 and Bacillus , and the fungal genera of Millerozyma , Penicillium , Mortierella , Monascus , Saccharomyces , Issatchenkia , Pithoascus , Pseudallescheria , and Wickerhamomyces. Additionally, we speculate that Sporosarcina is the predominant bacterial genus responsible for the imbalance of microbiota in pit mud. [ABSTRACT FROM AUTHOR]

Abstract Pit mud quality is a key parameter that impacts the quality of Chinese strong-flavor Baijiu production.This study was developed to explore spatial bacterial community distributions and the relationships between these distributions and the volatile compound accumulation within the pit mud used in the production of Chinese strong-flavor Baijiu. The results revealed Lactobacillus pasteurii and Limnochorda pilosa were found to be the dominant bacteria present in the upper wall, middle wall, and bottom pit mud layers, whereas the Clostridium genus was detectable at high levels in the lower layer of the pit wall and played a role in contributing to the overall aroma and flavor compounds in produced Chinese strong-flavor Baijiu, with Clostridium abundance being strongly correlated with caproic acid, ethyl caproate, ethyl butyrate, and hexanol levels as well as moderately correlated with butyric acid levels. The abundance of the Lactobacillus genus was positively correlated with levels of ethyl lactate, 1-butanol, and 2,3-butanediol. Limnochorda pilosa was closely associated with ethyl acetate levels. Additionally, the correlations between bacterial communities and chemical properties also investigated, and the results demonstrated PO43−, total carbon, K+, humus, NH4 +-N, and Mg2+ levels significantly affected the bacterial community structure of pit mud, and they were positively correlated with the relative abundance of Clostridium. Together, these findings can serve as a foundation for future studies exploring the mechanisms whereby volatile compounds accumulate in different pit mud layers, which facilitates the fermentation regulation and pit mud quality improvement of Chinese strong-flavor Baijiu.

Objective: The correlation degree between organic acids and microbial community structure of pit mud (PM) with different depths in new PM (3 years old) and old PM (30 years old) was analyzed in order to understand the aging and maturation rule of PM in vertical dimension. Methods: Illumina MiSeq high-throughput sequencing was used to analyze the succession of microbial community structure in PM, and the interaction between organic acids and microorganisms was analyzed by correlation analysis. Results: There was a significant difference between the deep (4~10 cm) and shallow (0~4 cm) layers of pit mud in the vertical dimension. Caproiciproducens occupied the dominant position in shallow pit mud of old pit cellar, while Fastidiosipila and Hydrogenispora occupied the dominant position in deep pit mud. In the new pit mud, Lactobacillus played a dominant role in both shallow and deep pit mud. The relative abundance of Bacillus and Clostridium sensu stricto 12 increased with the increase of pit mud depth. The shallow pit mud was positively correlated with water, acetic acid, hexanoic acid, lactic acid, butyric acid and humus, while the deep pit mud was positively correlated with NH4+-N, available phosphorus and pH. Correlation analysis of organic acids and microorganisms showed that Caloramatoraceae was positively correlated with organic acid synthesis in the old pit cellar. Lactobacillus and Hydrogenispora in the new pit were positively correlated with organic acid synthesis. Caproiciproducens was positively correlated with organic acid content in new and old cellars. Conclusion: The results of this study can clarify the prokaryotic community structure and physicochemical factors of vertical dimension of pit mud in the bottom of new and old pit mud, and provide reference for guiding the maturation and stability of pit mud.

Pit mud (PM) is the main component of Baijiu (traditional Chinese liquor), and its microorganisms are the primary sources of the aroma of Chinese strong-flavor Baijiu (SFB). Enrichment plays an important role in the selection of functional microorganisms in PM. Herein, the PM of SFB was submitted to six rounds of enrichment using clostridial growth medium (CGM), and changes in the metabolite accumulation and microbiota composition were evaluated. Based on the metabolite production and microbiota composition, the enrichment rounds were classified as the acclimation stage (round 2), main fermentation stage (rounds 3 and 4), and late fermentation stage (rounds 5 and 6). Species within the genus Clostridium dominated in the acclimation stage (65.84–74.51%). In the main fermentation stage, the dominant microbial groups were producers of butyric acid, acetic acid, and caproic acid, which included Clostridium (45.99–74.80%), Caproicibacter (1.45–17.02%), and potential new species within the order of Oscillataceae (14.26–29.10%). In the late stage of enrichment, Pediococcus dominated (45.96–79.44%). Thus, the main fermentation stage can be considered optimal for the isolation of acid-producing bacteria from PM. The findings discussed herein support the development and application of functional bacteria by bioaugmentation, and contribute to improving the quality of PM and SFB production. [ABSTRACT FROM AUTHOR]

Pit mud quality is a key parameter that impacts the quality of Chinese strong-flavor Baijiu production.This study was developed to explore spatial bacterial community distributions and the relationships between these distributions and the volatile compound accumulation within the pit mud used in the production of Chinese strong-flavor Baijiu. The results revealed Lactobacillus pasteurii and Limnochorda pilosa were found to be the dominant bacteria present in the upper wall, middle wall, and bottom pit mud layers, whereas the Clostridium genus was detectable at high levels in the lower layer of the pit wall and played a role in contributing to the overall aroma and flavor compounds in produced Chinese strong-flavor Baijiu, with Clostridium abundance being strongly correlated with caproic acid, ethyl caproate, ethyl butyrate, and hexanol levels as well as moderately correlated with butyric acid levels. The abundance of the Lactobacillus genus was positively correlated with levels of ethyl lactate, 1-butanol, and 2,3-butanediol. Limnochorda pilosa was closely associated with ethyl acetate levels. Additionally, the correlations between bacterial communities and chemical properties also investigated, and the results demonstrated PO43−, total carbon, K+, humus, NH4+-N, and Mg2+ levels significantly affected the bacterial community structure of pit mud, and they were positively correlated with the relative abundance of Clostridium. Together, these findings can serve as a foundation for future studies exploring the mechanisms whereby volatile compounds accumulate in different pit mud layers, which facilitates the fermentation regulation and pit mud quality improvement of Chinese strong-flavor Baijiu. [ABSTRACT FROM AUTHOR]

Abstract Background Chinese strong-flavor baijiu (CSFB), one of the three major baijiu types, is the most popular baijiu type among consumers in China. A variety of microbes are involved in metabolizing raw materials to produce ethanol and flavor substances during fermentation, which fundamentally determined the quality of baijiu. It is of great importance to study microbial community of fermented grains (zaopei) during baijiu brewing process for improving its quality. In this study, we firstly used propidium monoazide (PMA) to treat zaopei samples from 5-year pit and 20-year pit for removing the interference of non-viable fungi, and analyzed the diversity of total fungi and viable fungi by quantitative PCR (qPCR) and high-throughput sequencing (HTS) based on ITS2 gene. Results The results showed that total fungi and viable fungi displayed no significant differences at OTU, phylum, or genus levels during fermentation within two kinds of pits. A total of 6 phyla, 19 classes, and 118 genera in fungi were found based on OTUs annotation in zaopei samples from 5-year pit and 20-year pit. Besides, non-viable fungi had little effect on the fungal community diversity during the fermentation cycle. It was found that the most dominant viable fungi belonged to Saccharomyces, Kazachstania, Naumovozyma, and Trichosporon, and Naumovozyma was firstly detected in zaopei samples of CSFB. Moreover, based on the variation of flavor substances in zaopei samples, the quality of CSFB produced from older pit was better than that produced from younger pit. Conclusion The non-viable fungi had little effect on the fungal diversity, structure, and relative abundance in zaopei samples of CSFB, and Naumovozyma was firstly detected in zaopei samples of CSFB. Our findings can be applied as guidance for improving the quality and stability of CSFB.

Pit mud (PM) is the main component of Baijiu (traditional Chinese liquor), and its microorganisms are the primary sources of the aroma of Chinese strong-flavor Baijiu (SFB). Enrichment plays an important role in the selection of functional microorganisms in PM. Herein, the PM of SFB was submitted to six rounds of enrichment using clostridial growth medium (CGM), and changes in the metabolite accumulation and microbiota composition were evaluated. Based on the metabolite production and microbiota composition, the enrichment rounds were classified as the acclimation stage (round 2), main fermentation stage (rounds 3 and 4), and late fermentation stage (rounds 5 and 6). Species within the genus Clostridium dominated in the acclimation stage (65.84–74.51%). In the main fermentation stage, the dominant microbial groups were producers of butyric acid, acetic acid, and caproic acid, which included Clostridium (45.99–74.80%), Caproicibacter (1.45–17.02%), and potential new species within the order of Oscillataceae (14.26–29.10%). In the late stage of enrichment, Pediococcus dominated (45.96–79.44%). Thus, the main fermentation stage can be considered optimal for the isolation of acid-producing bacteria from PM. The findings discussed herein support the development and application of functional bacteria by bioaugmentation, and contribute to improving the quality of PM and SFB production.

Microorganisms in pit mud are the essential factor determining the style of strong flavor Baijiu. The spatial distribution characteristics of fungal communities and aroma in the pit mud for strong flavor Baijiu from Xinjiang, China, were investigated using Illumina MiSeq high-throughput sequencing and electronic nose technology. A total of 138 fungal genera affiliated with 10 fungal phyla were identified from 27 pit mud samples; of these, Saccharomycopsis, Aspergillus, and Apiotrichum were the core fungal communities, and Aspergillus and Apiotrichum were the hubs that maintain the structural stability of fungal communities in pit mud. The fungal richness and diversity, as well as aroma of pit mud, showed no significant spatial heterogeneity, but divergences in pit mud at different depths were mainly in pH, total acid, and high abundance fungi. Moisture, NH4+, and lactate were the main physicochemical factors involved in the maintenance of fungal stability and quality in pit mud, whereas pH had only a weak effect on fungi in pit mud. In addition, the fungal communities of pit mud were not significantly associated with the aroma. The results of this study provide a foundation for exploring the functional microorganisms and dissecting the brewing mechanism of strong flavor Baijiu in Xinjiang, and also contributes to the improvement of pit mud quality by bioaugmentation and controlling environmental physicochemical factors.

Microorganisms in pit mud are the essential factor determining the style of strong flavor Baijiu. The spatial distribution characteristics of fungal communities and aroma in the pit mud for strong flavor Baijiu from Xinjiang, China, were investigated using Illumina MiSeq high-throughput sequencing and electronic nose technology. A total of 138 fungal genera affiliated with 10 fungal phyla were identified from 27 pit mud samples; of these, Saccharomycopsis , Aspergillus , and Apiotrichum were the core fungal communities, and Aspergillus and Apiotrichum were the hubs that maintain the structural stability of fungal communities in pit mud. The fungal richness and diversity, as well as aroma of pit mud, showed no significant spatial heterogeneity, but divergences in pit mud at different depths were mainly in pH, total acid, and high abundance fungi. Moisture, NH4+, and lactate were the main physicochemical factors involved in the maintenance of fungal stability and quality in pit mud, whereas pH had only a weak effect on fungi in pit mud. In addition, the fungal communities of pit mud were not significantly associated with the aroma. The results of this study provide a foundation for exploring the functional microorganisms and dissecting the brewing mechanism of strong flavor Baijiu in Xinjiang, and also contributes to the improvement of pit mud quality by bioaugmentation and controlling environmental physicochemical factors. [ABSTRACT FROM AUTHOR]

Background: Chinese strong-flavor baijiu (CSFB), one of the three major baijiu types, is the most popular baijiu type among consumers in China. A variety of microbes are involved in metabolizing raw materials to produce ethanol and flavor substances during fermentation, which fundamentally determined the quality of baijiu. It is of great importance to study microbial community of fermented grains (zaopei) during baijiu brewing process for improving its quality. In this study, we firstly used propidium monoazide (PMA) to treat zaopei samples from 5-year pit and 20-year pit for removing the interference of non-viable fungi, and analyzed the diversity of total fungi and viable fungi by quantitative PCR (qPCR) and high-throughput sequencing (HTS) based on ITS2 gene. Results: The results showed that total fungi and viable fungi displayed no significant differences at OTU, phylum, or genus levels during fermentation within two kinds of pits. A total of 6 phyla, 19 classes, and 118 genera in fungi were found based on OTUs annotation in zaopei samples from 5-year pit and 20-year pit. Besides, non-viable fungi had little effect on the fungal community diversity during the fermentation cycle. It was found that the most dominant viable fungi belonged to Saccharomyces, Kazachstania, Naumovozyma, and Trichosporon, and Naumovozyma was firstly detected in zaopei samples of CSFB. Moreover, based on the variation of flavor substances in zaopei samples, the quality of CSFB produced from older pit was better than that produced from younger pit. Conclusion: The non-viable fungi had little effect on the fungal diversity, structure, and relative abundance in zaopei samples of CSFB, and Naumovozyma was firstly detected in zaopei samples of CSFB. Our findings can be applied as guidance for improving the quality and stability of CSFB. [ABSTRACT FROM AUTHOR]

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 log10 [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. [ABSTRACT FROM AUTHOR]

Pit mud microbial consortia play crucial roles in the formation of Chinese strong-flavor baijiu's key flavor-active compounds, especially butyric and caproic acids. Clostridia , one of the abundant bacterial groups in pit mud, were recognized as important butyric and caproic acid producers. Research on the interactions of the pit mud microbial community mainly depends on correlation analysis at present. Interaction between Clostridium and other microorganisms and its involvement in short/medium-chain fatty acid (S/MCFA) metabolism are still unclear. We previously found coculture of two clostridial strains isolated from pit mud, Clostridium fermenticellae JN500901 ( C. 901) and Novisyntrophococcus fermenticellae JN500902 ( N. 902), could enhance S/MCFA accumulation. Here, we investigated their underlying interaction mechanism through the combined analysis of phenotype, genome, and transcriptome. Compared to monocultures, coculture of C. 901 and N. 902 obviously promoted their growth, including shortening the growth lag phase and increasing biomass, and the accumulation of butyric acid and caproic acid. The slight effects of inoculation ratio and continuous passage on the growth and metabolism of coculture indicated the relative stability of their interaction. Transwell coculture and transcriptome analysis showed the interaction between C. 901 and N. 902 was accomplished by metabolite exchange, i.e., formic acid produced by C. 901 activated the Wood-Ljungdahl pathway of N. 902, thereby enhancing its production of acetic acid, which was further converted to butyric acid and caproic acid by C. 901 through reverse β-oxidation. This work demonstrates the potential roles of mutually beneficial interspecies interactions in the accumulation of key flavor compounds in pit mud. IMPORTANCE Microbial interactions played crucial roles in influencing the assembly, stability, and function of the microbial community. The metabolites of pit mud microbiota are the key to flavor formation of Chinese strong-flavor baijiu. So far, researches on the interactions of the pit mud microbial community have been mainly based on the correlation analysis of sequencing data, and more work needs to be performed to unveil the complicated interaction patterns. Here, we identified a material exchange-based mutualistic interaction system involving two fatty acid-producing clostridial strains ( Clostridium fermenticellae JN500901 and Novisyntrophococcus fermenticellae JN500902) isolated from pit mud and systematically elucidated their interaction mechanism for promoting the production of butyric acid and caproic acid, the key flavor-active compounds of baijiu. Our findings provide a new perspective for understanding the complicated interactions of pit mud microorganisms.

Daqu provides a complex and unique microbial ecosystem and acts as a fermentation starter and a source of flavor compounds for Chinese strong-flavor Baijiu production. Dynamic changes in Daqu bacterial composition and physicochemical properties during production were studied using physicochemical analysis and Illumina HiSeq sequencing, revealing that the bacterial α- and β-diversity changed markedly after the start of fermentation. The dominant bacterial genera were Cyanobacteria, Proteobacteria and Firmicutes; Enterobacter, within Proteobacteria, was at the most influential node of the interaction network, and was positively correlated with Bacillus , Pantoea and Pediococcus. Redundancy analysis showed that the bacterial community structure was closely related to the physicochemical properties of Daqu , especially the acidity, cellulase activity and moisture. These findings clarified the complex bacterial community succession and interactions between organisms and with physicochemical properties, during Daqu fermentation, thereby enabling quality improvement of strong-flavor Baijiu, by augmenting selected bacterial cultures, or by controlling environmental factors. [Display omitted] • Dynamics of bacterial community in strong-flavor liquor Daqu were detected. • Bacterial α- and β-diversity was significantly different between Day 0 and others. • The dominant bacterial taxa were Cyanobacteria, Proteobacteria, and Firmicutes. • Enterobacter was the keystone node of the bacterial network in Daqu. • Bacterial community structure was closely related to acidity, cellulase and moisture. [ABSTRACT FROM AUTHOR]