Characterizing the Contribution of Functional Microbiota Cultures in Pit Mud to the Metabolite Profiles of Fermented Grains.

Elevating the flavor profile of strong flavors Baijiu has always been a focal point in the industry, and pit mud (PM) serves as a crucial flavor contributor in the fermentation process of the fermented grains (FG). This study investigated the influence of wheat flour and bran (MC and FC) as PM culture enrichment media on the microbiota and metabolites of FG, aiming to inform strategies for improving strong-flavor Baijiu flavor. Results showed that adding PM cultures to FG significantly altered its properties: FC enhanced starch degradation to 51.46% and elevated reducing sugar content to 1.60%, while MC increased acidity to 2.11 mmol/10 g. PM cultures also elevated FG's ester content, with increases of 0.36 times for MC-FG60d and 1.48 times for FC-FG60d compared to controls, and ethyl hexanoate rising by 0.91 times and 1.39 times, respectively. Microbial analysis revealed that Lactobacillus constituted over 95% of the Abundant bacteria community, with Kroppenstedtia or Bacillus being predominant among Rare bacteria. Abundant fungi included Rasamsonia, Pichia, and Thermomyces, while Rare fungi consisted of Rhizopus and Malassezia. Metagenomic analysis revealed bacterial dominance, primarily consisting of Lactobacillus and Acetilactobacillus (98.80-99.40%), with metabolic function predictions highlighting genes related to metabolism, especially in MC-FG60d. Predictions from PICRUSt2 suggested control over starch, cellulose degradation, and the TCA cycle by fungal subgroups, while Abundant fungi and bacteria regulated ethanol and lactic acid production. This study highlights the importance of PM cultures in the fermentation process of FG, which is significant for brewing high-quality, strong-flavor Baijiu.

Revealing microbiota characteristics and predicting flavor-producing sub-communities in Nongxiangxing baijiu pit mud through metagenomic analysis and metabolic modeling.

The microorganisms of the pit mud (PM) of Nongxiangxing baijiu (NXXB) have an important role in the synthesis of flavor substances, and they determine attributes and quality of baijiu. Herein, we utilize metagenomics and genome-scale metabolic models (GSMMs) to investigate the microbial composition, metabolic functions in PM microbiota, as well as to identify microorganisms and communities linked to flavor compounds. Metagenomic data revealed that the most prevalent assembly of bacteria and archaea was Proteiniphilum, Caproicibacterium, Petrimonas, Lactobacillus, Clostridium, Aminobacterium, Syntrophomonas, Methanobacterium, Methanoculleus, and Methanosarcina. The important enzymes ofPMwere in bothGH and GT familymetabolism. A total of 38 high-quality metagenome-assembled genomes (MAGs) were obtained, including those at the family level (n = 13), genus level (n = 17), and species level (n = 8). GSMMs of the 38 MAGs were then constructed. From the GSMMs, individual and community capabilities respectively were predicted to be able to produce 111 metabolites and 598 metabolites. Twenty-three predicted metabolites were consistent with the metabonomics detected flavors and served as targets. Twelve sub-community of were screened by cross-feeding of 38 GSMMs. Of them, Methanobacterium, Sphaerochaeta, Muricomes intestini, Methanobacteriaceae, Synergistaceae, and Caloramator were core microorganisms for targets in each sub-community. Overall, this study of metagenomic and target-community screening could help our understanding of the metabolite-microbiome association and further bioregulation of baijiu.

Illuminating the Characteristics and Assembly of Prokaryotic Communities across a pH Gradient in Pit Muds for the Production of Chinese Strong-Flavor Baijiu.

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 ß-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.

Analysis of the differences in physicochemical properties, volatile compounds, and microbial community structure of pit mud in different time spaces.

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.

Resuscitation of baijiu pit mud bacteria based on Rpf protein of Umezawaea beigongshangensis.

The pit mud in the Baijiu fermentation cellar is an abundant microbial resource that is closely related to the quality of baijiu. However, many naturally existing species might be in a viable but nonculturable (VBNC) state, posing challenges to the isolation and application of functional species. Herein, a previously isolated strain from baijiu mash, Umezawaea beigongshangensis, was found to contain the rpf gene that encodes resuscitation promotion factor (Rpf). Therefore, the gene was cloned and heterologously expressed, and the recombinant protein (Ub-Rpf 2) was purified. Ub-Rpf 2 was found to significantly promote the growth of resuscitated VBNC state Corynebacterium beijingensis and Sphingomonas beigongshangensis. To determine the resuscitation effect of Ub-Rpf 2 on real ecological samples, the protein was supplemented in pit mud for enrichment culture. Results revealed that specific families and genera were enriched in abundance upon Ub-Rpf 2 incubation, including a new family of Symbiobacteraceae and culturable Symbiobacterium genus. Furthermore, 14 species belonging to 12 genera were obtained in Ub-Rpf 2 treated samples, including a suspected novel species. This study lays a foundation for applying Rpf from U. beigongshangensis to exploit microbial resources in baijiu pit mud.

Analysis of differences in microorganisms and aroma profiles between normal and off-flavor pit mud in Chinese strong-flavor Baijiu.

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.

Comparison of the Correlations of Microbial Community and Volatile Compounds between Pit-Mud and Fermented Grains of Compound-Flavor Baijiu.

The microbial composition and volatile components of fermented grains (FG) and pit mud (PM) are crucial for the quality and flavor of compound-flavor baijiu (CFB). The physicochemical indices, culturable microorganisms, microbial communities, and volatile components of FG and PM were analyzed and correlated in our research. Considering FG and PM, amplicon sequencing was used to analyze the microbial community and the volatile components were detected by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME). For FG, redundancy analysis and correlation perfume Circos were used to clarify the correlations between the dominant microbial community and volatile components. The results showed that Aspergillus, Pichia, and Rhizopus were the main fungal microflora in FG and PM, whereas Lactobacillus and Bacillus were the dominant bacteria in FG, and Methanosarcina and Clostridium sensu stricto 12 were the dominant bacteria in the PM. The microbial community and volatile compounds in the CB sampled from the bottom layers of the FG were greatly affected by those in the PM. There were 32 common volatile components in CB and PM. For FG, most of the volatile components were highly correlated with Lactobacillus, Bacillus, Aspergillus, Pichia, and Monascus, which includes alcohols, acids and esters. This study reveals correlations between microbial composition, volatile components, and the interplay of FG and PM, which are conducive to optimizing the fermentation process and improving the quality of CFB base.

Contribution of microorganisms from pit mud to volatile flavor compound synthesis in fermented grains for nongxiangxing baijiu brewing.

BACKGROUND: Nongxiangxing baijiu (NB) is known for its distinct flavor profile, which is attributed to key aroma compounds. The exposed fermentation technique, utilizing daqu and solid-state fermentation in pit muds, plays a crucial role in flavor development. Though previous studies have investigated the impact of microorganisms from pit ?ud and fermented grains on flavor compound production, a comprehensive understanding of microbial functions in the entire pit fermentation system is lacking. Herein, we aimed to explore the role of pit-mud-derived microorganisms in shaping the microbial community and flavor compound synthesis in NB. RESULTS: There are 76 volatile flavor compounds that have been identified in fermented grains during NB fermentation. The main flavor compounds in NB clustered within the same network module, and 27.27% of microorganisms in the core modules of the fermented grain co-occurrence network originated from pit mud. The relationship between pit mud microorganisms and flavor compounds revealed a significant positive correlation (92%). Notably, Prevotella and Sarocladium were identified as the main contributors to this effect on flavor. CONCLUSION: Microorganisms originating from pit mud influenced the composition and activity of microorganisms in fermented grains and facilitated the production of flavor compounds in NB. © 2023 Society of Chemical Industry.

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

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

Difference Analysis of the Composition of Iron (Hydr)Oxides and Dissolved Organic Matter in Pit Mud of Different Pit Ages in Luzhou Laojiao and Its Implications for the Ripening Process of Pit Mud.

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.

Microbial biogeography of pit mud from an artificial brewing ecosystem on a large time scale: all roads lead to Rome.

IMPORTANCE: Baijiu is a typical example of how humans employ microorganisms to convert grains into new flavors. Mud cellars are used as the fermentation vessel for strong-flavor Baijiu (SFB) to complete the decomposition process of grains. The typical flavor of SFB is mainly attributed to the metabolites of the pit mud microbiome. China has a large number of SFB-producing regions. Previous research revealed the temporal profiles of the pit mud microbiome in different geographical regions. However, each single independent study rarely yields a thorough understanding of the pit mud ecosystem. Will the pit mud microbial communities in different production regions exhibit similar succession patterns and structures under the impact of the brewing environment? Hence, we conducted research in pit mud microbial biogeography to uncover the impact of specific environment on the microbial community over a long time scale.

Succession of microbial community of the pit mud under the impact of Daqu of Nongxiang Baijiu.

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 H2 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.

Genome Analysis and Optimization of Caproic Acid Production of Clostridium butyricum GD1-1 Isolated from the Pit Mud of Nongxiangxing Baijiu.

Caproic acid is a precursor substance for the synthesis of ethyl caproate, the main flavor substance of nongxiangxing baijiu liquor. In this study, Clostridium butyricum GD1-1, a strain with high caproic acid concentration (3.86 g/l), was isolated from the storage pit mud of nongxiangxing baijiu for sequencing and analysis. The strain's genome was 3,840,048 bp in length with 4,050 open reading frames. In addition, virulence factor annotation analysis showed C. butyricum GD1-1 to be safe at the genetic level. However, the annotation results using the Kyoto Encyclopedia of Genes and Genomes Automatic Annotation Server predicted a deficiency in the strain's synthesis of alanine, methionine, and biotin. These results were confirmed by essential nutrient factor validation experiments. Furthermore, the optimized medium conditions for caproic acid concentration by strain GD1-1 were (g/l): glucose 30, NaCl 5, yeast extract 10, peptone 10, beef paste 10, sodium acetate 11, L-cysteine 0.6, biotin 0.004, starch 2, and 2.0% ethanol. The optimized fermentation conditions for caproic acid production by C. butyricum GD1-1 on a single-factor basis were: 5% inoculum volume, 35°C, pH 7, and 90% loading volume. Under optimal conditions, the caproic acid concentration of strain GD1-1 reached 5.42 g/l, which was 1.40 times higher than the initial concentration. C. butyricum GD1-1 could be further used in caproic acid production, NXXB pit mud strengthening and maintenance, and artificial pit mud preparation.

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

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

Yeast diversity in pit mud and related volatile compounds in fermented grains of chinese strong-flavour liquor.

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.

Bacterial Communities Found in Pit-Wall Mud and Factors Driving Their Evolution.

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.

Effect of Multiple Rounds of Enrichment on Metabolite Accumulation and Microbiota Composition of Pit Mud for Baijiu Fermentation.

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.

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

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

Comparing the differences of prokaryotic microbial community between pit walls and bottom from Chinese liquor revealed by 16S rRNA gene sequencing.

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).

Bacterial diversity associated with volatile compound accumulation in pit mud of Chinese strong-flavor baijiu pit.

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.