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Background: Pit mud (PM) hosts diverse microbial communities, which serve as a medium to impart flavor and quality to Baijiu and exhibit long-term tolerance to ethanol and acids, resulting in a unique ecosystem. However, the ecology and metabolic functions of PM remain poorly understood, as many taxa in PM represent largely novel lineages. In this study, we used a combination of metagenomic analysis and chemical derivatization LC–MS analysis to provide a comprehensive overview of microbial community structure, metabolic function, phylogeny, horizontal gene transfer, and the relationship with carboxyl compounds in spatiotemporal PM samples. Results: Our findings revealed three distinct stages in the spatiotemporal changes of prokaryotic communities in PM: an initial phase dominated by Lactobacillus, a transitional phase, and a final state of equilibrium. Significant variations in α- and β-diversity were observed across different spatial and temporal PM samples. We identified 178 medium- and high-quality non-redundant metagenome-assembled genomes (MAGs), and constructed their phylogenetic tree, depicting their roles in the carbon, nitrogen, and sulfur cycles. The Wood-Ljungdahl pathway and reverse TCA cycle were identified as the main carbon fixation mechanisms, with both hydrogenotrophic and aceticlastic methanogens playing a major role in methane production, and methylotrophic pathway observed in older PM. Furthermore, we identified relationships between prokaryotes and 29 carboxyl metabolites, including medium- and long-chain fatty acids. Horizontal gene transfer (HGT) was widespread in PM, particularly among clostridia, Bacteroidota, Bacilli, and Euryarchaeota, and was shown to play critical roles in fermentation dynamics, carbon fixation, methane production, and nitrogen and sulfur metabolism. Conclusion: Our study provides new insights into the evolution and function of spatiotemporal PM, as well as its interactions with carboxyl metabolites. Lactobacillus dominated in new PM, while methanogens and clostridia were predominant in older or deeper PM layers. The three distinct stages of prokaryotic community development in PM and HGT played critical roles in metabolic function of spatiotemporal PM. Furthermore, this study highlights the importance of α-diversity, β-diversity, methanogens, and Clostridium as useful indicators for assessing PM quality in the production of high-quality Baijiu. [ABSTRACT FROM AUTHOR]

Solid-state fermentation in mud pits is a typical brewing process for nongxiangxing baijiu and fuyuxiangxing baijiu. Pit mud is the functional carrier of mud pits, in which the abundance, diversity, metabolic activity and interaction of microorganisms could directly affect the quality of baijiu. This paper reviews the application of traditional culture and non-culture techniques in research on microorganisms in pit mud, the spatiotemporal differences in microbial community structure, the functional microorganisms and their interactions, the development and improvement of the methods for the quality evaluation of pit mud, and pit mud maintenance and aging focusing on the functional strains, and summarizes the development history and application status of different techniques in the field of research on microorganisms related to the production of Chinese baijiu, the growth and decline patterns of prokaryotic and eukaryotic microorganisms during pit mud aging, the spatial difference in the distribution of prokaryotic and eukaryotic microorganisms in the pit, and the functions and interdependence of the core functional microorganisms in pit mud such as caproic acid bacteria, butyric acid bacteria, lactic acid bacteria, acetic acid bacteria and methane bacteria, the reason for pit mud aging, the transformation of the criteria for quality evaluation pit mud from traditional sensory and physicochemical analysis to the comprehensive evaluation system, and studies on pit mud maintenance and aging based on the functional properties of core strains. The purpose of this review is to provide a reference for evaluating the microbial diversity, exploring the metabolic function of microbial community and improving the quality of pit mud.

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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]

Pit mud (PM), as an important source of microorganisms, is necessary for Chinese strong-flavor baijiu (CSFB) production. Although it has been revealed that the PM prokaryotic community diversities are influenced by its quality, product area, ages, etc., the characteristics and assembly process of the prokaryotic community in PMs across a pH gradient are still unclear. In this study, the regular changes of α- and β-diversities of the prokaryotic community across a pH gradient in PMs were revealed, which could be divided into "stable", "relatively stable", and "drastically changed" periods. A total of 27 phyla, 53 classes, and 381 genera were observed in all given samples, dominated by Firmicutes, Bacteroidetes, Proteobacteria, Lactobacillus, Caproiciproducens, Proteiniphilum, etc. Meanwhile, the complexity of the network structure of the prokaryotic microbial communities is significantly influenced by pH. The community assembly was jointly shaped by deterministic and stochastic processes, with stochastic process contributing more. This study was a specialized report on elucidating the characteristics and assembly of PM prokaryotic communities across a pH gradient, and revealed that the diversity and structure of PM prokaryotic communities could be predictable, to some degree, which could contribute to expanding our understanding of prokaryotic communities in PM. [ABSTRACT FROM AUTHOR]

The fungal community structure, the relationship between fungal flora and physicochemical factors, and the prediction of fungal function in pit muds from different spatial positions of 10- and 50-year-old cellars at Jinhui liquor Co. Ltd. were studied by using Illumina NovaSeq high-throughput sequencing, redundancy analysis and Fungi Functional Guild (FUNGuild). The results showed that the fungal diversity and richness of the 10-year-old pit mud decreased with increasing depth; the fungal diversity of the 50-year-old pit mud showed an overall increasing trend, while the fungal richness initially decrease and then increased. Moreover, for the 10-year-old pit, the fungal diversity and richness of the upper layer of the pit wall were significantly higher than those of the other positions (P < 0.05), while for the 50-year-old cellar, the fungal diversity and richness of the bottom layer were significantly higher than those of the other locations (P < 0.05). The fungal diversity and richness were significantly higher in the wall of the 10-year-old cellar than the 50-year-old cellar (P < 0.05), but were significantly higher in the bottom of the 50-year-old cellar than the 10-year-old cellar (P < 0.05). A total of 21 fungal phyla and 520 genera were detected in all pit mud samples, the relative abundance of four dominant phyla (Ascomycota, Basidiomycota, Mortierellomycota and Rozellomycota) and most dominant genera such as Aspergillus and Kazachstania showed significant changes among pit ages and spatial locations (P < 0.05). Fusarium, Aspergillus, Saccharomyces and Monascus were positively correlated with the contents of water, humus, K+ and Ca2+, while Cladosporium and Vishniacozyma were positively correlated with pH. Seven nutritional modes of fungi were observed, mainly including saprophytic and pathological-saprophytic-symbiotic nutritional modes, and four single and seven mixed functional groups were determined. This study provides a theoretical basis for clarifying the structure and spatial distribution of fungal community in Jinhui Baijiu pit mud.

In this study, high-throughput sequencing technology, linear discriminant analysis effect size (LEfSe) and redundancy analysis (RDA) were used to investigate the structure of prokaryotic and eukaryotic communities, the differential microbial communities, and the relationship between microorganisms and physicochemical factors in pit mud (PM) from cellars of different ages (5, 10 and 20 years) and from different spatial locations of cellars. The results showed that the diversity of both prokaryotic and eukaryotic microorganisms was higher in the five-year-old PM than in the 10- and 20-year-old PM. Among all samples, a total of 37 phyla, 83 classes, 176 orders, 306 families and 629 genera of prokaryotic microorganisms were detected, with five phyla being the dominant ones. Totally 12 phyla, 37 orders, 74 families, 160 families and 282 genera of eukaryotic microorganisms were detected, including three phyla and 15 genera being dominant. LEfSe identified 12 differential prokaryotic genera and 15 differential eukaryotic species, and the differential microbial communities of each sample played an important role in the fermentation process of baijiu. The results of physicochemical analysis showed that the pH of the PM samples varied from 3.66 to 5.20, and the effective phosphorus content of PM was significantly different among locations. RDA analysis showed that Lactobacillus, unclassified_f__Aspergillaceae, and Thermomyces were negatively correlated with moisture, ammonia nitrogen, and effective phosphorus contents, and Methanobacterium, Clostridium_sensu_stricto_12, Caproiciproducens, Methanobrevibacter, Cryptococcus_f__Tremellaceae and Apiotrichum were positively correlated with moisture, pH, ammonia nitrogen, and effective phosphorus. This study provides theoretical support for the establishment of daily PM maintenance as well as high-quality PM domestication and production.

Pit mud (PM) is among the key factors determining the quality of Nongxiangxing baijiu, a Chinese liquor. Microorganisms present inside PM are crucial for the unique taste and flavor of this liquor. In this study, headspace solid-phase microextraction was used in combination with gas chromatography and high-throughput sequencing to determine the volatile compounds and microbial community structure of 10- and 40-year PM samples from different spaces. The basic physicochemical properties of the PM were also determined. LEfSe and RDA were used to systematically study the PM in different time spaces. The physicochemical properties and ester content of the 40-year PM were higher than those of the 10-year PM, but the spatial distribution of the two years PM samples exhibited no consistency, except in terms of pH, available phosphorus content, and ester content. In all samples, 29 phyla, 276 families, and 540 genera of bacteria, including four dominant phyla and 20 dominant genera, as well as eight phyla, 24 families, and 34 genera of archaea, including four dominant phyla and seven dominant genera, were identified. The LEfSe analysis yielded 18 differential bacteria and five differential archaea. According to the RDA, the physicochemical properties and ethyl caproate, ethyl octanoate, hexanoic acid, and octanoic acid positively correlated with the differential microorganisms of the 40-year PM, whereas negatively correlated with the differential microorganisms of the 10-year PM. Thus, we inferred that Caproiciproducens, norank_f__Caloramatoraceae, and Methanobrevibacter play a dominant and indispensable role in the PM. This study systematically unveils the differences that affect the quality of PM in different time spaces and offers a theoretical basis for improving the declining PM, promoting PM aging, maintaining cellars, and cultivating an artificial PM at a later stage. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Chinese strong-flavour liquor is produced via a traditional solid-state fermentation strategy facilitated by live microorganisms in pit mud-based cellars. For the present analysis, pit mud samples from different spatial locations within fermentation cellars were collected, and the yeast communities therein were assessed via culture-based and denaturing gradient gel electrophoresis (DGGE) approaches. These analyses revealed significant differences in the composition of yeast communities present in different layers of pit mud. In total, 29 different yeast species were detected, and principal component analyses revealed clear differences in microbial diversity in pit mud samples taken from different cellar locations. Culture-dependent strategies similarly detected 20 different yeast species in these samples. However, while Geotrichum silvicola, Torulaspora delbrueckii, Hanseniaspora uvarum, Saturnispora silvae, Issatchenkia orientalis, Candida mucifera, Kazachstania barnettii, Cyberlindnera jadinii, Hanseniaspora spp., Alternaria tenuissima, Cryptococcus laurentii, Metschnikowia spp., and Rhodotorula dairenensis were detected via a PCR-DGGE approach, they were not detectable in culture-dependent analyses. In contrast, culture-based approaches led to the identification of Schizosaccharomyces pombe and Debaryomyces hansenii in these pit mud samples, whereas they were not detected using DGGE fingerprints profiles. An additional HS-SPME-GC-MS-based analysis of the volatile compounds present in fermented grains samples led to the identification of 66 such compounds, with the highest levels of volatile acids, esters, and alcohols being detected in fermented grains from lower layer samples. A canonical correspondence analysis (CCA) suggested they were significant correlations between pit mud yeast communities and associated volatile compounds in fermented grains.

Long-term production practice proves that good liquor comes out of the old cellar, and the aged pit mud is very important to the quality of Luzhou-flavor liquor. X-ray diffraction, Fourier transform ion cyclotron resonance mass spectrometry, and infrared spectroscopy were used to investigate the composition characteristics of iron-bearing minerals and dissolved organic matter (DOM) in 2-year, 40-year, and 100-year pit mud and yellow soil (raw materials for making pit mud) of Luzhou Laojiao distillery. The results showed that the contents of total iron and crystalline iron minerals decreased significantly, while the ratio of Fe(II)/Fe(III) and the content of amorphous iron (hydr)oxides increased significantly with increasing cellar age. DOM richness, unsaturation, and aromaticity, as well as lignin/phenolics, polyphenols, and polycyclic aromatics ratios, were enhanced in pit mud. The results of the principal component analysis indicate that changes in the morphology and content of iron-bearing minerals in pit mud were significantly correlated with the changes in DOM molecular components, which is mainly attributed to the different affinities of amorphous iron (hydr)oxides and crystalline iron minerals for the DOM components. The study is important for understanding the evolution pattern of iron-bearing minerals and DOM and their interactions during the aging of pit mud and provides a new way to further understand the influence of aged pit mud on Luzhou-flavor liquor production. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Complex microbiomes of pit mud play significant roles in imbuing flavors and qualities of Nongxiang Baijiu during fermentation. However, pit mud microbial enrichment and succession is a long process that is also accompanied by aging. Development of high-quality artificial pit mud becomes an urgent problem. In this study, a new medium based on space (TK) Daqu was used to effectively enrich the dominant microorganisms in pit mud. The results showed that Caproiciproducens was the most preponderance in the cultures unadded Daqu , whereas Clostridium sensu stricto 12 was the most preponderance, followed by Caproiciproducens in the enrichment cultures added TK Daqu. It is worth noting that TK Daqu balanced the relative abundance of Caproiciproducens and Clostridium sensu stricto 12 in 100-year pit mud culture (S100), which was more conducive to the increase of methanogens. PICRUSt2 prediction results showed that hydrogenotrophic methanogens could promote the synthesis of caproic acid by using the product H 2 as the metabolic substrate and increased significantly in the pit mud enrichment cultures with TK Daqu. The increase of lactate dehydrogenase (EC 1.1.1.27) content in S100 contributed to the degradation of lactic acid and the increase of caproic acid. Adding TK Daqu enrichment cultures is more conducive to the enrichment and metabolic balance of pit mud microorganisms. [Display omitted] • TK Daqu was used to influence pit mud microbiotas. • TK Daqu was beneficial to the enrichment of Clostridium and methanogenic. • TK Daqu facilitates the increase of caproic acid and decrease of lactic acid. [ABSTRACT FROM AUTHOR]

Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used for the detection of terpenoids in the raw materials, Daqu, pit mud, fermented grains and base liquor during the brewing process of Nongxiangxing baijiu. Totally 19 terpenoids were detected from the nine raw materials, and their total concentration was 1 338.22 μg/kg. In addition, 18 terpenoids were detected in Daqu at eight time points during fermentation with a total concentration of 2 020.32 μg/kg. A total of 13 terpenoids were detected in pit mud from 10 different cellars with a total concentration of 2 167.89 μg/kg. Eight terpenoids were detected in fermented grains at four time points with a total concentration of 702.68 μg/kg. Nine terpenoids were detected in two groups of base liquor with a total concentration of 338.56 μg/kg. Comparative analysis showed that two of the nine terpenoids came from the raw materials, three came from Daqu, and two were converted from other substances due to high temperatures during the steaming process.

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.

AbstractThe richness and diversity of bacterial community in the fermented pit mud from the new and old distilleries of Shedian Liquor were analysed via the Illumina Miseq PE300 high-throughput sequencing technology, so as to further understand the differences of bacterial community structure in fermented pit muds with different pit ages. The results show that the bacterial community is mainly distributed into five dominant phyla (> 1.0%): Firmicutes, Synergistes, Bacteroidetes, Proteobacteria and Verrucomicrobia. There are 13 dominant genera (> 1.0%), of which Lactobacillus is dominant in both samples with a higher content in the new pit (61.12%) and lower in the old pit (1.15%). Caproiciproducens accounting for 43.53% and Syntrophomonas accounting for 6.20%, norank_f__Family_XIV (17.68%), Aminobacterium (17.68%), Sedimentibacter (1.89%), Caldicoprobacter (1.52%) and Sporanaerobacter (1.29%) are the dominant bacteria in the old pit mud. Bacillus accounting for 6.02%, Bacteroides (6.27%), Akkermansia (1.51%), Hydrogenispora (2.15%) and norank_f__Bacteroidales_S24-7_group (1.15%) are the dominant bacteria in the new pit mud. The results showed that there is a great difference in the bacterial community structure of in the new and old pit mud. In addition, compared with the bacterial community composition in the pit mud of Luzhou flavour Baijiu in Sichuan, it was found that the microorganisms that formed caproic acid and butyric acid were different.

ABSTRACT The fermentation process of strong-flavor Baijiu occurs in an underground cellar surrounded by mud, where grains are decomposed and generate new flavor substances. Pit mud microbiome plays a pivotal role in the formation of Baijiu flavor, which is positively correlated with cellar age. Previous research on the pit mud microbiome was often conducted for a specific distillery, making it hard to draw a general conclusion to decipher the pit mud microecosystem in such a unique brewing environment. Hence, in this study, we attempt to unravel community formation mechanisms through research in pit mud microbial biogeography based on 12 independent bacterial studies comprising qualified data sets of 302 samples clearly marked with cellar age from five major producing provinces. We found cellar age explained more variations in the bacterial community than geographical region. The bacterial community displayed evident dominant species alternations on a large time scale. Lactobacillus was found to be the dominant species in young pit mud (

Chinese strong-flavour liquor is produced via a traditional solid-state fermentation strategy facilitated by live microorganisms in pit mud-based cellars. For the present analysis, pit mud samples from different spatial locations within fermentation cellars were collected, and the yeast communities therein were assessed via culture-based and denaturing gradient gel electrophoresis (DGGE) approaches. These analyses revealed significant differences in the composition of yeast communities present in different layers of pit mud. In total, 29 different yeast species were detected, and principal component analyses revealed clear differences in microbial diversity in pit mud samples taken from different cellar locations. Culture-dependent strategies similarly detected 20 different yeast species in these samples. However, while Geotrichum silvicola, Torulaspora delbrueckii, Hanseniaspora uvarum, Saturnispora silvae, Issatchenkia orientalis, Candida mucifera, Kazachstania barnettii, Cyberlindnera jadinii, Hanseniaspora spp., Alternaria tenuissima, Cryptococcus laurentii, Metschnikowia spp., and Rhodotorula dairenensis were detected via a PCR-DGGE approach, they were not detectable in culture-dependent analyses. In contrast, culture-based approaches led to the identification of Schizosaccharomyces pombe and Debaryomyces hansenii in these pit mud samples, whereas they were not detected using DGGE fingerprints profiles. An additional HS-SPME-GC-MS-based analysis of the volatile compounds present in fermented grains samples led to the identification of 66 such compounds, with the highest levels of volatile acids, esters, and alcohols being detected in fermented grains from lower layer samples. A canonical correspondence analysis (CCA) suggested they were significant correlations between pit mud yeast communities and associated volatile compounds in fermented grains. [ABSTRACT FROM AUTHOR]

Pit mud (PM), as an important source of microorganisms, is necessary for Chinese strong-flavor baijiu (CSFB) production. Although it has been revealed that the PM prokaryotic community diversities are influenced by its quality, product area, ages, etc., the characteristics and assembly process of the prokaryotic community in PMs across a pH gradient are still unclear. In this study, the regular changes of α- and β-diversities of the prokaryotic community across a pH gradient in PMs were revealed, which could be divided into “stable”, “relatively stable”, and “drastically changed“ periods. A total of 27 phyla, 53 classes, and 381 genera were observed in all given samples, dominated by Firmicutes, Bacteroidetes, Proteobacteria, Lactobacillus, Caproiciproducens, Proteiniphilum, etc. Meanwhile, the complexity of the network structure of the prokaryotic microbial communities is significantly influenced by pH. The community assembly was jointly shaped by deterministic and stochastic processes, with stochastic process contributing more. This study was a specialized report on elucidating the characteristics and assembly of PM prokaryotic communities across a pH gradient, and revealed that the diversity and structure of PM prokaryotic communities could be predictable, to some degree, which could contribute to expanding our understanding of prokaryotic communities in PM.

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-wall mud (PWM) fosters bacterial communities involved in Baijiu production. PWM varies depending on pit age and height. In this study, we explored the bacterial communities in PWM and factors driving their evolution. The abundance and diversity of bacterial communities were low in new PWM (NPWM). In old PWM (OPWM), similar but diverse bacterial communities were observed at different heights. Lactobacillus was the predominant genus in NPWM, and Caproiciproducens, Aminobacterium, Hydrogenispora, Lactobacillus, Petrimonas, Syntrophomonas, and Sedimentibacter were the dominant genera in OPWM. A decrease was noted in the abundance of Lactobacillus, which indicated evolution. Among all the physicochemical properties, pH had the highest degree of interpretation with an R2 value of 0.965. pH also exerted the strongest effect on bacterial communities. The path coefficients of pH on bacterial community diversity and abundance were 0.886 and 0.810, respectively. Caproiciproducens and Clostridium sensu stricto 12 metabolized lactic acid, inhibiting the growth of Lactobacillus at a suitable pH, which led to the maturation of PWM. Our findings enrich the literature on the evolution of bacterial communities in PM and the maturation of PM. [ABSTRACT FROM AUTHOR]

Copyright of Shipin Kexue/ Food Science is the property of Food Science Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Abstract Purpose The microbial community in the pit mud correlated closely with the quality of the final product of Chinese strong-flavored Baijiu (CSFB). However, environmental conditions and brewing processes can vary by region and distilleries. This may lead to differences in microbial composition and function in pit mud. Therefore, revealing the features of the pit mud microbial community structure and functions of different distilleries will provide key information for understanding the diversity and difference of microbes in the brewing of CSFB, which will be beneficial for the improvement of the quality of pit mud and CSFB in the future. Methods and results Illumina MiSeq sequencing of 16S rRNA gene amplicons was used to analyze the similarities and differences in microbial community structure and function in pit muds of different distilleries located in Shihezi (Xinjiang), Xiangyang (Hubei), and Yibin (Sichuan). At the genus level, Clostridium, Lactobacillus, Aminobacterium, Petrimonas, Syntrophomonas, Methanoculleus, Syntrophaceticus, Sedimentibacter, Caloramator, Ruminococcus, Bacillus, Methanosarcina, and Garciella were the dominated genera of pit muds. There were great differences in the composition of microorganisms in pit muds used by different distilleries. The significantly enriched prokaryotic microbiotas of pit muds collected in the distilleries of Xiangyang were mainly affiliated with Bacillus, Lactobacillus, and Croceifilum, and the relative abundance of methanogens, such as Methanomicrobia and Methanobacteria, were only significantly enriched in the pit mud collected from the distilleries of Yibin (P < 0.05). Functional analysis indicated that the difference of microbial composition in pit mud will further lead to significant differences in various metabolic functions. Conclusion The compositions and functions of dominant microorganisms in pit mud used for the production of CSFB by different enterprises across regions in China were greatly different, and there was a close relationship between the compositions and functions of microorganisms in pit mud. Therefore, it may be an effective method to improve CSFB fermentation processes by directionally regulating the microbial community functions of pit mud using specific strains.

In order to study the spatial distribution of microorganisms and volatile compounds in pit mud, solid phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to qualitatively and quantitatively analyze the main volatile compounds in pit mud, and Illumina MiSeq sequencing was used to analyze the composition of microbial community in pit mud. The results of the study showed that the volatile components in the pit mud were mainly esters and acids, accounting for 73.01% to 98.36%, and they continued to increase with the increase of the depth of the pit. A total of 10 phyla, 15 classes, 21 orders, 42 families, and 64 genera of prokaryotic microorganisms were detected in the pit mud samples. The dominant flora accounted for 98.86%~99.93% of the total number of bacteria at the phylum level, and Firmicutes was the absolute dominant flora. The Shannon index increased from 2.130 to 4.970, the Simpson index increased from 0.557 to 0.932, and the ACE index and Chao1 index were the highest at the bottom of the pool, at 114.523 and 114.50, respectively. Results of α-diversity indicated that the abundance and diversity of microbial flora in the lower part of the pool wall and pit mud at the bottom of the pool were higher than that in the upper and middle parts of the pool wall, which was consistent with the type and content distribution of volatile substances. Dominant bacteria such as Clostridium and Methanobacter were the main microorganisms which could carry out microbial metabolism and inter-species material exchange, and produce important aroma substances and their precursors. As the depth of space increased, they continued to react and accumulate, closely related to spatial location of the mud. Spatial difference was an important reason for the differences in the composition of prokaryotic microbes and volatile substances in different spatial locations of pit mud.

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]

This study aims to explore the prokaryotic microbial community structures and diversity in pit mud from different depths, and provide a theoretical basis for the liquor production and further study of pit mud. The fermented pit muds of strong-flavor liquor from Yun distillery were taken as samples. The high-throughput sequencing approach, followed by bioinformatics analyses, was used to compare the differences in the prokaryotic microbial community between pit walls and bottom represented by samples. A total of 31 bacteria phyla and 2 archaea phyla were detected. The dominant phyla in YJ-S, YJ-Z, and YJ-X (sample name) were Proteobacteria and Firmicutes, while the dominant genera in them were Acinetobacter, Aminobacterium, and Lactobacillus. YJ-Z and YJ-X were the closest in species diversity. In species richness analysis, YJ-X was the highest, followed by YJ-Z, and YJ-S was the lowest; in species uniformity analysis, YJ-S was the highest, followed by YJ-Z, and YJ-X was the lowest. The function predicted by 16S rRNA genome showed that prokaryotic microbial function in pit mud was mainly concentrated in “Carbohydrate transport and metabolism” and “Amino acid transport and metabolism.” Significant differences in prokaryotic microbial community and gene function prediction between pit walls and bottom were found in YJ-S, YJ-Z, and YJ-X (p < 0.05). [ABSTRACT FROM AUTHOR]

Purpose: The microbial community in the pit mud correlated closely with the quality of the final product of Chinese strong-flavored Baijiu (CSFB). However, environmental conditions and brewing processes can vary by region and distilleries. This may lead to differences in microbial composition and function in pit mud. Therefore, revealing the features of the pit mud microbial community structure and functions of different distilleries will provide key information for understanding the diversity and difference of microbes in the brewing of CSFB, which will be beneficial for the improvement of the quality of pit mud and CSFB in the future. Methods and results: Illumina MiSeq sequencing of 16S rRNA gene amplicons was used to analyze the similarities and differences in microbial community structure and function in pit muds of different distilleries located in Shihezi (Xinjiang), Xiangyang (Hubei), and Yibin (Sichuan). At the genus level, Clostridium, Lactobacillus, Aminobacterium, Petrimonas, Syntrophomonas, Methanoculleus, Syntrophaceticus, Sedimentibacter, Caloramator, Ruminococcus, Bacillus, Methanosarcina, and Garciella were the dominated genera of pit muds. There were great differences in the composition of microorganisms in pit muds used by different distilleries. The significantly enriched prokaryotic microbiotas of pit muds collected in the distilleries of Xiangyang were mainly affiliated with Bacillus, Lactobacillus, and Croceifilum, and the relative abundance of methanogens, such as Methanomicrobia and Methanobacteria, were only significantly enriched in the pit mud collected from the distilleries of Yibin (P < 0.05). Functional analysis indicated that the difference of microbial composition in pit mud will further lead to significant differences in various metabolic functions. Conclusion: The compositions and functions of dominant microorganisms in pit mud used for the production of CSFB by different enterprises across regions in China were greatly different, and there was a close relationship between the compositions and functions of microorganisms in pit mud. Therefore, it may be an effective method to improve CSFB fermentation processes by directionally regulating the microbial community functions of pit mud using specific strains. [ABSTRACT FROM AUTHOR]

Long-term production practice proves that good liquor comes out of the old cellar, and the aged pit mud is very important to the quality of Luzhou-flavor liquor. X-ray diffraction, Fourier transform ion cyclotron resonance mass spectrometry, and infrared spectroscopy were used to investigate the composition characteristics of iron-bearing minerals and dissolved organic matter (DOM) in 2-year, 40-year, and 100-year pit mud and yellow soil (raw materials for making pit mud) of Luzhou Laojiao distillery. The results showed that the contents of total iron and crystalline iron minerals decreased significantly, while the ratio of Fe(II)/Fe(III) and the content of amorphous iron (hydr)oxides increased significantly with increasing cellar age. DOM richness, unsaturation, and aromaticity, as well as lignin/phenolics, polyphenols, and polycyclic aromatics ratios, were enhanced in pit mud. The results of the principal component analysis indicate that changes in the morphology and content of iron-bearing minerals in pit mud were significantly correlated with the changes in DOM molecular components, which is mainly attributed to the different affinities of amorphous iron (hydr)oxides and crystalline iron minerals for the DOM components. The study is important for understanding the evolution pattern of iron-bearing minerals and DOM and their interactions during the aging of pit mud and provides a new way to further understand the influence of aged pit mud on Luzhou-flavor liquor production.

Clostridium is the key bacteria that inhabits the pit mud in a fermentation cell, for the production of strong-flavor Baijiu. Its activities in the process of Baijiu fermentation is closely related to the niches of pit mud and cells. After multiple rounds of underground fermentation, Clostridium has been domesticated and adapted to the environment. The mechanisms of clostridia succession in the pit mud and how they metabolize nutrients present in grains are not clear. In this study, 15 Clostridium species including three firstly reported ones (Clostridium tertium, Clostridium pabulibutyricum and Clostridium intestinale) in strong-flavor Baijiu pit mud, were isolated from the pit mud. Eighty one percent of these Clostridium strains are motile, and most of them show chemotaxis to organic acids, glutathione, saccharides and lactic acid bacteria. In a simulated Baijiu fermentation system, Clostridium migrated from pit mud to fermented grains with the addition of chemokine lactic acid, resulting in the production of acetic acid and butyric acid. The results help to understand the succession mechanism of Clostridium in pit mud, and provide a reference for regulation of lactic acid level in fermented grains during Baijiu fermentation. [ABSTRACT FROM AUTHOR]

Actinomycetes (a group of filamentous bacteria) are the dominant microbial order in the Daqu (DQ) fermentation starter and in the pit mud (PM) of the Baijiu fermentation microbiome. Actinomycetes produce many of the key enzymes and flavor components, and supply important precursors, which have a major influence on its characteristic aroma components, to other microorganisms during fermentation. This paper reviews the current progress on actinomycete research related to Baijiu fermentation, including the isolation and identification, distribution, interspecies interactions, systems biology, and main metabolites. The main metabolites and applications of the actinomycetes during Baijiu fermentation are also discussed. [ABSTRACT FROM AUTHOR]

Pit mud, a specific fermented soil, is an essential material for the fermentation of Chinese strong-flavour liquor. However, few studies to date have sought to characterize the spatial profiles of pit mud fungal communities in fermentation cellars from Chinese strong-flavour liquor distilleries. In this analysis, differences in fungal community structures and physicochemical properties in pit mud samples from different spatial positions within fermentation cellars were analyzed, revealing unique characteristic multidimensional pit mud fungal community profiles. Penicillium roqueforti, Pichia kudriavzevii, Aotearoamyces nothofagi, Penicillium robsamsonii, Alternaria arborescens, Trichosporon insectorum, Seltsamia ulmi, Trichosporon coremiiforme, Malassezia restricta were dominant in the pit mud samples form the upper cellar wall, whereas Metarhizium frigidum, Calonectria pseudoreteaudii, Penicillium clavigerum, Fusarium equiseti, Simplicillium chinense, Aspergillus intermedius, Trichosporon coremiiforme, Fusarium circinatum, Alternaria radicina, Aspergillus heterocaryoticus were predominant in the middle cellar wall. Alternaria radicina, Cladosporium chasmanthicola, Alternaria helianthiinficiens, Penicillium argentinense, Antarctomyces psychrotrophicus, and Trichosporon inkin are majorly present in the down cellar wall layer. Bipolaris axonopicola, Ramgea ozimecii, Penicillium argentinense, Calonectria queenslandica, Metarhizium robertsii, and Penicillium roqueforti were identified as the dominant fungi in pit mud samples from the cellar bottom. Additionally, Alternaria destruens and Alternaria doliconidium are present at notably high levels in all layers of pit mud samples. Moisture, pH, PO43−, acetic acid, humus, K+, Mg2+, Ca2+, butyric acid, and caproic acid levels in these different pit mud positions exhibited a rising incremental pattern from the upper wall layer to the bottom layer, whereas lactic acid levels were significantly lower in the bottom pit mud layer relative to these other layers. Moisture, pH, and NH4+-N were identified as the three most significant factors associated with fungal community composition through a redundancy analysis. Overall, these findings may offer a theoretical foundation for future efforts to improve or standardize artificial pit mud. [ABSTRACT FROM AUTHOR]

Caproic acid is the precursor of ethyl caproate, the main representative flavor substance of strong-flavor baijiu (SFB). Caproic acid-producing bacteria are considered to be the most important type of acid-producing microorganisms in the pit mud of the SFB ecosystem. In this study, the Rummeliibacillus suwonensis 3B-1 with a high yield of caproic acid (4.064 g/L) was screened from SFB pit mud. The genome of the R. suwonensis 3B-1 was sequenced, the total size was found to be 4117,671 bp and a calculated GC content of 35.86%. The caproic acid biosynthesis pathway was identified and analyzed, and it showed that 3B-1 could not only use ethanol, but it could also use glucose and other carbon sources as substrates to produce caproic acid. According to the genome analysis and with an optimized medium, the optimal conditions for caproic acid production were yeast powder at 3 g/L, sodium acetate at 15 g/L, and 1% biotin at 8 mL/100 mL. The yield of caproic acid reached 4.627 g/L, an increase of 13.9%, which was higher than that of general caproic acid bacteria. This is the first report of the synthesis of caproic acid by R. suwonensis. This strain could be used to produce caproic acid, an artificial pit mud preparation, and/or an enhanced inoculum in the production of SFB. [Display omitted] • Rummeliibacillus suwonensis was reported as the caproic acid producing bacteriu-m for the first time. • Caproic acid biosynthetic pathway of Rummeliibacillus suwonensis was identified. • The yield of caproic acid reached 4.627 g/L based on the pathway analysis and fermentation medium optimization. [ABSTRACT FROM AUTHOR]

ABSTRACT 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.

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Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Background: Although studies have shown that Bacteroidetes, Clostridiales, and Lactobacillales are the main components of the microbial community in pit mud during the brewing of Luzhou-flavored liquor, little is known about the effect of brewing materials on spatial structures of this microbiome. Methods: High-throughput sequencing of the V4-V5 region of prokaryotic 16S rRNA gene was performed to analyze the microbial community diversity and spatial heterogeneity in Luzhou-flavored liquor pit muds with different brewing ingredients. The structural characteristics and heterogeneous spatial distribution of the pit mud microbial communities were examined using bioinformatics and multivariate statistical analysis methods. Results: Our results showed that Euryarchaeota, Actinobacteria, Bacteroidetes, Chlorobi, Chloroflexi, Firmicutes, Proteobacteria, Synergistetes, Tenericutes, and WWE1 were the dominant phyla in the pit mud microbiome. The Shannon and Simpson indices of the pit mud microbiome with three grains (M3G) in the upper layer were significantly lower than those in middle layer and bottom, whereas those of the pit mud microbiome with five grains (M5G) in bottom were significantly lower than those in middle layer (p < 0.05). There were significant differences in the microbial community compositions between the pit muds with different brewing ingredients and locations in the same pit (p < 0.05). T78 of Anaerolinaceae, Butyrivibrio, Dehalobacter_Syntrophobotulus, Desulfosporosinus, Asteroleplasma, and vadinCA02 of Synergistaceae were significantly enriched in M3G, whereas Prevotella, Vagococcus, Caldicoprobacter, Butyrivibrio, Coprococcus, Dorea, Sporanaerobacter, Tepidimicrobium, TissierellaSoehngenia, RFN20 of Erysipelotrichaceae, Sutterella, 125ds10 of Alteromonadales, Vibrio, and Sphaerochaeta were significantly enriched in M5G. This study provides a theoretical basis for exploring the influence of brewing ingredients in pit muds on the production of Luzhou-flavored liquor and the specific influence of pit mud microorganisms in different locations on liquor production. [ABSTRACT FROM AUTHOR]

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.

Pit-wall mud (PWM) fosters bacterial communities involved in Baijiu production. PWM varies depending on pit age and height. In this study, we explored the bacterial communities in PWM and factors driving their evolution. The abundance and diversity of bacterial communities were low in new PWM (NPWM). In old PWM (OPWM), similar but diverse bacterial communities were observed at different heights. Lactobacillus was the predominant genus in NPWM, and Caproiciproducens, Aminobacterium, Hydrogenispora, Lactobacillus, Petrimonas, Syntrophomonas, and Sedimentibacter were the dominant genera in OPWM. A decrease was noted in the abundance of Lactobacillus, which indicated evolution. Among all the physicochemical properties, pH had the highest degree of interpretation with an R2 value of 0.965. pH also exerted the strongest effect on bacterial communities. The path coefficients of pH on bacterial community diversity and abundance were 0.886 and 0.810, respectively. Caproiciproducens and Clostridium sensu stricto 12 metabolized lactic acid, inhibiting the growth of Lactobacillus at a suitable pH, which led to the maturation of PWM. Our findings enrich the literature on the evolution of bacterial communities in PM and the maturation of PM.

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]

The richness and diversity of bacterial community in the fermented pit mud from the new and old distilleries of Shedian Liquor were analysed via the Illumina Miseq PE300 high-throughput sequencing technology, so as to further understand the differences of bacterial community structure in fermented pit muds with different pit ages. The results show that the bacterial community is mainly distributed into five dominant phyla (> 1.0%): Firmicutes, Synergistes, Bacteroidetes, Proteobacteria and Verrucomicrobia. There are 13 dominant genera (> 1.0%), of which Lactobacillus is dominant in both samples with a higher content in the new pit (61.12%) and lower in the old pit (1.15%). Caproiciproducens accounting for 43.53% and Syntrophomonas accounting for 6.20%, norank_f__Family_XIV (17.68%), Aminobacterium (17.68%), Sedimentibacter (1.89%), Caldicoprobacter (1.52%) and Sporanaerobacter (1.29%) are the dominant bacteria in the old pit mud. Bacillus accounting for 6.02%, Bacteroides (6.27%), Akkermansia (1.51%), Hydrogenispora (2.15%) and norank_f__Bacteroidales_S24-7_group (1.15%) are the dominant bacteria in the new pit mud. The results showed that there is a great difference in the bacterial community structure of in the new and old pit mud. In addition, compared with the bacterial community composition in the pit mud of Luzhou flavour Baijiu in Sichuan, it was found that the microorganisms that formed caproic acid and butyric acid were different. [ABSTRACT FROM AUTHOR]

This study aims to explore the prokaryotic microbial community structures and diversity in pit mud from different depths, and provide a theoretical basis for the liquor production and further study of pit mud. The fermented pit muds of strong-flavor liquor from Yun distillery were taken as samples. The high-throughput sequencing approach, followed by bioinformatics analyses, was used to compare the differences in the prokaryotic microbial community between pit walls and bottom represented by samples. A total of 31 bacteria phyla and 2 archaea phyla were detected. The dominant phyla in YJ-S, YJ-Z, and YJ-X (sample name) were Proteobacteria and Firmicutes, while the dominant genera in them were Acinetobacter, Aminobacterium, and Lactobacillus. YJ-Z and YJ-X were the closest in species diversity. In species richness analysis, YJ-X was the highest, followed by YJ-Z, and YJ-S was the lowest; in species uniformity analysis, YJ-S was the highest, followed by YJ-Z, and YJ-X was the lowest. The function predicted by 16S rRNA genome showed that prokaryotic microbial function in pit mud was mainly concentrated in “Carbohydrate transport and metabolism” and “Amino acid transport and metabolism.” Significant differences in prokaryotic microbial community and gene function prediction between pit walls and bottom were found in YJ-S, YJ-Z, and YJ-X (p < 0.05).

Clostridium beijerinckii is a well-known anaerobic solventogenic bacterium which inhabits a wide range of different niches. Previously, we isolated five butyrate-producing C. beijerinckii strains from pit mud (PM) of strong-flavor baijiu (SFB) ecosystems. Genome annotation of the five strains showed that they could assimilate various carbon sources as well as ammonium to produce acetate, butyrate, lactate, hydrogen, and esters but did not produce the undesirable flavors isopropanol and acetone, making them useful for further exploration in SFB production. Our analysis of the genomes of an additional 233 C. beijerinckii strains revealed an open pangenome based on current sampling and will likely change with additional genomes. The core genome, accessory genome, and strain-specific genes comprised 1567, 8851, and 2154 genes, respectively. A total of 298 genes were found only in the five C. beijerinckii strains from PM, among which only 77 genes were assigned to Clusters of Orthologous Genes categories. In addition, 15 transposase and 12 phage integrase families were found in all five C. beijerinckii strains from PM. Between 18 and 21 genome islands were predicted for the five C. beijerinckii genomes. The existence of a large number of mobile genetic elements indicated that the genomes of the five C. beijerinckii strains evolved with the loss or insertion of DNA fragments in the PM of SFB ecosystems. This study presents a genomic framework of C. beijerinckii strains from PM that could be used for genetic diversification studies and further exploration of these strains. [ABSTRACT FROM AUTHOR]

The transformation of diverse feedstocks into medium-chain fatty acids (MCFAs) by mixed cultures is a promising biorefinery route because of the high value of MCFAs. A particular concern is how to maintain the microbial consortia in mixed cultures to achieve stable MCFA production. The Chinese strong aroma-type liquor (Baijiu) fermentation system continually produces caproic acid for decades through a spontaneous inoculation of anaerobes from pit mud into fermented grains. Therefore, illuminating the dominant caproate-producing bacterium (CPB) in pit mud and how the CPB is sustained in the spontaneous fermentation system will help to reveal the microbiological mechanisms of stable caproate production. Here, we examined pit mud samples across four Chinese strong aroma-type Baijiu-producing areas and found that a caproate- producing Caproicibacterium sp. was widely distributed in these distilleries, with relative abundance ranging from 1.4 to 35.5% and an average abundance of 11.4%. Through controlling carbon source availability, we obtained different simplified caproate- producing consortia and found that the growth advantage of Caproicibacterium sp. was highly dependent on glucose. Then, two strains, named Caproicibacterium sp. strain LBM19010 and Caproicibacterium sp. strain JNU-WLY1368, were isolated from pit mud of two regions. The metabolic versatility of this species utilizing starch, maltose, glucose, and lactate reflected its adaptability to the fermentation environment where these carbon sources coexist. The simultaneous utilization of glucose and lactate contributed to the balance between cell growth and pH homeostasis. This study reveals that multiple adaptation strategies employed by the predominant CPB promotes its stability and dominance in a saccharide- and lactate-rich anaerobic habitat. [ABSTRACT FROM AUTHOR]

Pit mud, a specific fermented soil, is an essential material for the fermentation of Chinese strong-flavour liquor. However, few studies to date have sought to characterize the spatial profiles of pit mud fungal communities in fermentation cellars from Chinese strong-flavour liquor distilleries. In this analysis, differences in fungal community structures and physicochemical properties in pit mud samples from different spatial positions within fermentation cellars were analyzed, revealing unique characteristic multidimensional pit mud fungal community profiles. Penicillium roqueforti, Pichia kudriavzevii, Aotearoamyces nothofagi, Penicillium robsamsonii, Alternaria arborescens, Trichosporon insectorum, Seltsamia ulmi, Trichosporon coremiiforme, Malassezia restricta were dominant in the pit mud samples form the upper cellar wall, whereas Metarhizium frigidum, Calonectria pseudoreteaudii, Penicillium clavigerum, Fusarium equiseti, Simplicillium chinense, Aspergillus intermedius, Trichosporon coremiiforme, Fusarium circinatum, Alternaria radicina, Aspergillus heterocaryoticus were predominant in the middle cellar wall. Alternaria radicina, Cladosporium chasmanthicola, Alternaria helianthiinficiens, Penicillium argentinense, Antarctomyces psychrotrophicus, and Trichosporon inkin are majorly present in the down cellar wall layer. Bipolaris axonopicola, Ramgea ozimecii, Penicillium argentinense, Calonectria queenslandica, Metarhizium robertsii, and Penicillium roqueforti were identified as the dominant fungi in pit mud samples from the cellar bottom. Additionally, Alternaria destruens and Alternaria doliconidium are present at notably high levels in all layers of pit mud samples. Moisture, pH, PO43−, acetic acid, humus, K+, Mg2+, Ca2+, butyric acid, and caproic acid levels in these different pit mud positions exhibited a rising incremental pattern from the upper wall layer to the bottom layer, whereas lactic acid levels were significantly lower in the bottom pit mud layer relative to these other layers. Moisture, pH, and NH4+-N were identified as the three most significant factors associated with fungal community composition through a redundancy analysis. Overall, these findings may offer a theoretical foundation for future efforts to improve or standardize artificial pit mud.

Actinomycetes (a group of filamentous bacteria) are the dominant microbial order in the Daqu (DQ) fermentation starter and in the pit mud (PM) of the Baijiu fermentation microbiome. Actinomycetes produce many of the key enzymes and flavor components, and supply important precursors, which have a major influence on its characteristic aroma components, to other microorganisms during fermentation. This paper reviews the current progress on actinomycete research related to Baijiu fermentation, including the isolation and identification, distribution, interspecies interactions, systems biology, and main metabolites. The main metabolites and applications of the actinomycetes during Baijiu fermentation are also discussed.

Clostridium is the key bacteria that inhabits the pit mud in a fermentation cell, for the production of strong-flavor Baijiu. Its activities in the process of Baijiu fermentation is closely related to the niches of pit mud and cells. After multiple rounds of underground fermentation, Clostridium has been domesticated and adapted to the environment. The mechanisms of clostridia succession in the pit mud and how they metabolize nutrients present in grains are not clear. In this study, 15 Clostridium species including three firstly reported ones (Clostridium tertium, Clostridium pabulibutyricum and Clostridium intestinale) in strong-flavor Baijiu pit mud, were isolated from the pit mud. Eighty one percent of these Clostridium strains are motile, and most of them show chemotaxis to organic acids, glutathione, saccharides and lactic acid bacteria. In a simulated Baijiu fermentation system, Clostridium migrated from pit mud to fermented grains with the addition of chemokine lactic acid, resulting in the production of acetic acid and butyric acid. The results help to understand the succession mechanism of Clostridium in pit mud, and provide a reference for regulation of lactic acid level in fermented grains during Baijiu fermentation.

The Baijiu-making microbiota has an important role in the alcohol production, flavor, and character of Baijiu. 16S rRNA gene sequencing revolutionized the understanding of Baijiu-making microbiota. In this study, nine phyla, 23 classes, 49 orders, 99 families, and 201 genera were detected in pit muds (PMs) by 16S rRNA gene sequencing. Firmicutes and Bacteroidetes predominated (>99%). At the order level, Clostridiales, Bacteroidales, and Bacillales predominated (>92%). At the genus level, Hydrogenispora, Petrimonas, Proteiniphilum, and Sedimentibacter predominated. The pure culture of Baijiu-making prokaryotes was essential to elucidating the role of these microbes in the fermentation of Baijiu. According to the theory of microbial culturomics, a culturing approach with multiple culture conditions was adopted, combining 16S rRNA gene sequencing. We identified 215 prokaryotic strains, which were assigned to 66 species, 41 genera, four phyla, and 19 potential new species. Gas conditions were key factors in culturomics. In addition, culturomics significantly increased the number of species isolated from the fermentation PM compared with previous reports. With culturomics, the diversity spectrum of culturable bacteria in the PM was increased 273.33% at the genus level. This study confirms the complementary role of culturomics in the exploration of complex microbiota.

Microbiota in the pit mud (PM) plays a crucial role in the production of Chinese strong-flavor liquor (CSFL), the most popular distilled liquor in China. However, previous studies used total microbes, instead of viable ones, for the characterization of the microbial community in this environment. In this study, we used propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing to verify the effect of non-viablee bacteria on the characterization of PM bacteria. After PMA concentration optimization, 50 μM PMA was chosen to pretreat 5 and 20 years PMs. The qPCR results showed that there were 50.78 and 71.84% of non-viable bacteria in the 5-year PM and 20-year PM, respectively. Both copy numbers of total bacteria and viable bacteria were significantly higher in 20-year PM than those in 5-year PM. Nevertheless, in terms of bacterial diversity and composition analyses at the operational taxonomic unit (OTU), phylum, class, and genus levels, 16S rRNA gene sequencing results displayed no significant differences between total bacteria and viable bacteria in both PM types. In conclusion, it is necessary for non-viable bacteria to be considered in determining absolute biomass of bacteria in PM, but not necessary in the analysis of diversity and composition of PM bacteria. To the best of our knowledge, our study is the first attempt to analyze viable bacteria in the PM of CSFL and provides useful information on how to accurately characterize a microbial community in a PM environment.