Chinese Strong-flavor Baijiu Research Articles

Caproiciproducens converts lactic acid into caproic acid during Chinese strong-flavor Baijiu brewing

Strong-flavor Baijiu, a type of Chinese liquor, is produced through anaerobic solid-state fermentation in a sealed mud pit. Ethyl caproate, the characteristic flavor compound of strong-flavor Baijiu, is influenced by caproic acid-producing bacteria in the pit mud. To better understand the formation of caproic acid, this study investigated the microbial composition and physicochemical parameters of pit mud from different layers (top, middle, and bottom) in Hubei and Sichuan provinces, China. The results revealed that Caproiciproducens plays a key role in caproic acid production by using lactic acid as a substrate, with its abundance increasing with the depth of the pit mud. A strain Caproiciproducens sp. R1, isolated from the pit mud, was shown to produce caproic acid from lactic acid within an initial pH range of 5.5–9.0 and lactic acid concentrations of 1 %–5 % (m/v). In addition, inoculation of strain R1 into Huangshui (a lactic acid-rich liquid from Baijiu production) resulted in 40.72 mM caproic acid production. This study demonstrates that Caproiciproducens plays a crucial role in caproic acid production from lactic acid during the fermentation process of strong-flavor Baijiu.

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 CFSB 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. Indole, 3-methyl-, which have been proven to be responsible for the muddy and musty flavors, were detected in both types of pit mud, and for the first time, high levels of 4-Methylanisole were 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. While we speculate that Sporosarcina is the predominant bacterial genus responsible for the imbalance of microbiota in pit mud.

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 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.

Seasonal dynamics of the microbial community in a strong-flavor baijiu fermentation.

The microbial community plays a crucial role in Chinese strong-flavor baijiu (SFB) fermentation. However, the seasonal dynamics of the microbial community in the SFB fermentation system and its contribution to the unique flavor of SFB have not been fully elucidated. In this study, we investigated the seasonal dynamics of the microbial community through 16S rRNA and ITS gene sequencing. The results revealed significant temporal dynamics of microbial communities and environmental variables throughout the four seasons. The influence of seasons on fungal communities was found to be more significant than on bacterial communities. The diversity of bacteria was higher during the winter and summer, whereas fungal diversity was more prominent in summer and autumn. Stochastic processes maintained their dominance in microbial assembly throughout all four seasons but the significance of heterogeneous selection increased during summer for both bacteria and fungi, whereas homogeneous selection became more pronounced during winter for fungi. The pH and environmental temperature were important drivers of microbial community assembly across different seasons, primarily impacting the core genera responsible for the production of major volatile flavor compounds (VFCs), especially ethyl caproate. These findings provide new insights into the impact of seasons on microbial communities and hold promise for improving the quality-control measures for SFB brewed in different seasons. © 2024 Society of Chemical Industry.

Fungal biogeographical patterns are key drivers shaping the regional flavor profiles of Chinese strong-flavor Baijiu

Despite the observation of regionally distinct flavor characteristics of Chinese Strong-flavor Baijiu (CSFB) in recent decades, we still lack a comprehensive understanding of how such geography-dependent flavors establish. Here, 168 samples from 8 distilleries were collected across the main production regions of CSFB, and the factors driving the establishment of regional characteristics were investigated by amplicon and metatranscriptome sequencing combined with measurements of climate environments. We demonstrated that microbial communities in both fermented grains and surrounding environments (air and ground) exhibit regional patterns. More importantly, we found the regional patterns of microbial communities at the functional level by contributional diversity analysis. Such microbial and functional patterns correlated with the volatile metabolites profiles of finished fermented grains and predicted the abundance of metabolites in finished fermented grains. Furthermore, we found that climate factors, particularly mean relative humidity, affected fungal rather than bacterial diversity in fermented grains and environments (air and ground), thereby contributing to CSFB regional distinctiveness. Our study provided a basic landscape of the regional patterns of microbiota and metabolites across the main CSFB-producing regions and the dominating shaping factors.

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

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

Effect of 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.

Uncovering acid resistance genes in lactic acid bacteria and impact of non-viable bacteria on bacterial community during Chinese strong-flavor baijiu fermentation

Chinese strong-flavor baijiu (CSFB) brewing is a spontaneously solid-state fermentation process for approximately 60 days. Numerous microorganisms grow, die, and spark a series of metabolic reactions during fermentation. In this study, the microbial community and structure between total and viable bacteria in zaopei from the 5- and 20-year pits of CSFB are revealed by amplicon sequencing. Metagenome sequencing was applied to investigate acid resistance genes in Lactobacillus and predict carbohydrate active enzyme in zaopei. Besides, SourceTracker was conducted to expose bacterial sources. Results revealed that there was no significant difference in the bacterial community and structure between the total and viable bacteria; Lactobacillus was the most dominant bacterium in zaopei of two types of pits. Meanwhile, acid resistance genes argR, aspA, ilvE, gshA, DnaK, and cfa were genes that sustained Lactobacillus survival in the late stages of fermentation with high contents of acid and ethanol, and glycosyltransferases were identified as the predominated enzymes during the CSFB fermentation which catalyzed the process of lactic acid generation via Embden-Meyerhof-Parnas pathway and Hexose Monophosphate Pathway. Moreover, the environment contributed most bacteria to zaopei of the 5- and 20-year pits. These findings will provide a deeper understanding of the microbial community structure of viable and total bacteria and the reason for the dominance of Lactobacillus in the later stages of CSFB fermentation.

Stochastic Processes Drive the Assembly and Metabolite Profiles of Keystone Taxa during Chinese Strong-Flavor Baijiu Fermentation

Multispecies communities participate in the fermentation of Chinese strong-flavor Baijiu (CSFB), and the metabolic activity of the dominant and keystone taxa is key to the flavor quality of the final product. However, their roles in metabolic function and assembly processes are still not fully understood. Here, we identified the variations in the metabolic profiles of dominant and keystone taxa and characterized their community assembly using 16S rRNA and internal transcribed spacer (ITS) gene amplicon and metatranscriptome sequencing. We demonstrate that CSFB fermentations with distinct metabolic profiles display distinct microbial community compositions and microbial network complexities and stabilities. We then identified the dominant taxa (Limosilactobacillus fermentum, Kazachstania africana, Saccharomyces cerevisiae, and Pichia kudriavzevii) and the keystone ecological cluster (module 0, affiliated mainly with Thermoascus aurantiacus, Weissella confusa, and Aspergillus amstelodami) that cause changes in metabolic profiles. Moreover, we highlight that the alpha diversity of keystone taxa contributes to changes in metabolic profiles, whereas dominant taxa exert their influence on metabolic profiles by virtue of their relative abundance. Additionally, our results based on the normalized stochasticity ratio (NST) index and the neutral model revealed that stochastic and deterministic processes together shaped CSFB microbial community assemblies. Stochasticity and environmental selection structure the keystone and dominant taxa differently. This study provides new insights into understanding the relationships between microbial communities and their metabolic functions. IMPORTANCE From an ecological perspective, keystone taxa in microbial networks with high connectivity have crucial roles in community assembly and function. We used CSFB fermentation as a model system to study the ecological functions of dominant and keystone taxa at the metabolic level. We show that both dominant taxa (e.g., those taxa that have the highest relative abundances) and keystone taxa (e.g., those taxa with the most cooccurrences) affected the resulting flavor profiles. Moreover, our findings established that stochastic processes were dominant in shaping the communities of keystone taxa during CSFB fermentation. This result is striking as it suggests that although the controlled conditions in the fermentor can determine the dominant taxa, the uncontrolled rare keystone taxa in the microbial community can alter the resulting flavor profiles. This important insight is vital for the development of potential manipulation strategies to improve the quality of CSFB through the regulation of keystone species.

Exploration of seasonal fermentation differences and the possibility of flavor substances as regulatory factors in Daqu

Medium-high temperature Daqu is a characteristic starter for Chinese strong-flavor Baijiu fermentation, and its final quality determines the character and type of Baijiu. Nonetheless, its formation is affected by the interaction of physical and chemical, environmental and microbial interaction, and the differences in seasonal fermentation performance emerge. Here, the differences in the two seasons' Daqu fermentation properties were revealed by the detection of the enzyme activity. The respective dominant enzyme in summer Daqu (SUD) was protease and amylase, while cellulase and glucoamylase in spring Daqu (SPD). The underlying causes of this phenomenon were then investigated through an evaluation of nonbiological variables and microbial community structure. A greater absolute number of microorganisms, particularly Thermoactinomyces, were created in the SPD as a result of the superior growth environment (higher water activity). Additionally, the correlation network and discriminant analysis hypothesized that the volatile organic compound (VOC) guaiacol, which had a different content between SUD and SPD, may be a contributing element to the microbial composition. In contrast to SUD, the enzyme system activity related to guaiacol production in SPD was significantly higher. To support this notion that the volatile flavor chemicals mediate microbial interactions in Daqu, the growth effect of guaiacol on several bacteria isolated from the Daqu was examined in both a contact and non-contact manner. This study emphasized that VOCs not only have the basic characteristics of flavor compounds but also have ecological significance. Because the strains' varied structures and enzyme activities affected how the microorganisms interacted, the VOCs produced in this way ultimately had a synergistic effect on the various effects of Daqu fermentation.

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.

Isolation, Identification, and Fermentation Medium Optimization of a Caproic Acid‑Producing Enterococcus casseliflavus Strain from Pit Mud of Chinese Strong Flavor Baijiu Ecosystem.

Caproic acid is the precursor material of ethyl hexanoate, a representative flavor substance in strong flavor baijiu (SFB). Increasing the content of caproic acid in SFB helps to improve its quality. In the present study, caproic acid-producing bacteria from the pit mud of an SFB ecosystem were isolated, purified, and characterized. Strain BF-1 with the highest caproic acid yield (0.88 g/l) was selected. The morphological and molecular identification analysis showed that strain BF-1 was Enterococcus casseliflavus. The genome of E. casseliflavus BF-1 was sequenced and was found to be 2,968,377 bp in length with 3,270 open reading frames (ORFs). The caproic acid biosynthesis pathway in E. casseliflavus BF-1 was predicted based on the KAAS annotation. The virulence factors in the genome of strain BF-1 were annotated, which showed that E. casseliflavus BF-1 is safe at the genetic level. After adding essential nutrients based on the KAAS annotation, the optimum medium conditions for acid production by strain BF-1 were obtained by performing orthogonal experiments. The caproic acid yield of strain BF-1 reached 3.03 g/l, which was 3.44-fold higher than the initial yield. The optimized fer- mentation of caproic acid production by BF-1 was reported for the first time. The strain could be further used to regulate the ecosystem in baijiu production to improve its quality.

Analysis of Physicochemical Properties and Microbial Succession of Wenwang Chinese Strong-flavor Baijiu at Different Depths in Old and New Pit Muds

Analysis of Physicochemical Properties and Microbial Succession of Wenwang Chinese Strong-flavor Baijiu at Different Depths in Old and New Pit Muds

Effects of multiple cycles of sorghum starch gelatinization and fermentation on production of Chinese strong flavor Baijiu

The property changes of sorghum starch during multiple cycles of gelatinization and fermentation were studied. This study simulated the gelatinization and fermentation process of raw starch in strong-flavor Baijiu production (Sorghum as raw material, the distillers' grains as a control). The results showed that the starch content of the same batch of sorghum after five cycles of gelatinization and fermentation was 9.98% (Cannot continue to be used for fermentation), and about 60% of the starch was consumed in the first three cycles of gelatinization and fermentation. The gel properties of sorghum starch gradually decreased during fermentation but slightly increased after gelatinization. After five cycles of gelatinization and fermentation, the sorghum starch has a uniform size and a thin and small fragment structure. At the same time, sorghum starch does not form new groups. The recrystallization of starch caused by multiple cycles of gelatinization and fermentation increased the onset gelatinization temperature (from 61.6 to 114.4 °C), peak gelatinization temperature (from 78.5 to 139.5 °C), and gelatinization enthalpy (from 7.980 to 17.121 J/g) of sorghum starch. The crystalline structure of sorghum starch changed from the initial type A to type A + V, type A + B + V, and finally to type B.

Bioremediation of degraded pit mud by indigenous microbes for Baijiu production

Microbes in pit mud play key roles in fermentation cellars for Chinese strong-flavor Baijiu (SFB) production. Pit mud, however, is frequently degraded during production, compromising the quality of the end product. In this study, a bioremediation method was used to restore degraded pit mud (DPM) using indigenous microbes derived from SFB production. Metabolomics and metagenomics were used to determine the dynamics of prokaryotes during DPM restoration and their link to SFB production. The composition of flavor compounds in SFB changed (P = 0.0001) before and after restoration of DPM. Consistent with the improved sensory quality, the ethyl caproate/ethyl lactate ratio, an SFB quality measure, increased after restoration (P < 0.001). The concentrations of humus, NH4+, available phosphorus, and available potassium in DPM increased during the restoration process (P < 0.05), which is consistent with high-quality pit mud. The relative abundance of microbes that are beneficial to SFB fermentation, such as Caproiciproducens, a bacterium that produces caproic acid, increased during the restoration process. Furthermore, a total of 18 metabolic pathways were enriched (P < 0.05) from DPM before and after restoration. This includes butanoate metabolism and pyruvate metabolism, which are related to the synthesis of key flavor esters in SFB.

Effect of microbial interaction on flavor quality in Chinese baijiu fermentation.

Chinese baijiu brewing is an open, complex, and synergetic functional microbiota fermentation process. Microbial interaction is pivotal for the regulation of microbial structure and function in the brewing microecosystem, consequently affecting the flavor and quality of baijiu. This article mainly summarizes the effect of microbial interactions among functional microbiota on the growth performance, flavor formation, and safe quality of baijiu fermentation process. In addition, the review specifically emphasizes on the microbial interactions for the regulation of “Ethyl Caproate-Increasing and Ethyl Lactate-Decreasing” in Chinese strong-flavor baijiu. Furthermore, the construction of synthetic microbiota by metabolic characteristics of the functional microbes and their interactions for regulating and controlling flavor quality of Chinese baijiu is also reviewed and prospected.

Clostridium btbubcensis BJN0001, a potentially new species isolated from the cellar mud of Chinese strong-flavor baijiu, produces ethanol, acetic acid and butyric acid from glucose.

The online version contains supplementary material available at 10.1007/s13205-022-03271-7.

Comparative analysis of the microbiotas and physicochemical properties inside and outside medium-temperature Daqu during the fermentation and storage

Medium-temperature Daqu (MT-Daqu), a saccharification-fermentation agent and aroma-producing agent, is used to produce Chinese strong-flavor Baijiu. Many related studies have been published; however, less is known about microbial community and quality properties inside and outside the MT-Daqu from fermentation to storage. Here, along with determining the physicochemical index, the microbial community of MT-Daqu was investigated using both culture-dependent and culture-independent methods during 31 days of fermentation and 4 months of storage. Volatile compounds of mature MT-Daqu were analyzed using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC–MS). The results indicated obvious variation in the microbial community due to the changes in environmental conditions, and the physicochemical indices shifted from fluctuations in the fermentation period to relative stability after storage for 3 months. Moreover, the microbial counts and physicochemical indices of the inner layers of MT-Daqu differed from those of the outer layers. The dominant communities, including the bacterial phyla Firmicutes, Proteobacteria, and Actinobacteria and the fungal phyla Ascomycota and Mucoromycota, showed different abundances in the two parts of the mature MT-Daqu, and different microbial communities were enriched in both parts. Additionally, pyrazines and alcohols were the most abundant volatile aroma compounds in the mature MT-Daqu.

Identification and characterization of a novel tetrapeptide from enzymatic hydrolysates of Baijiu byproduct

In order to prepare angiotensin I-converting enzyme (ACE) inhibitory peptides, distilled spent grains of Chinese strong-flavor Baijiu were hydrolyzed by alcalase followed by papain under optimized conditions. A superior ACE inhibitory peptide was separated and purified by ultrafiltration and high-performance liquid chromatography (HPLC), and its amino acid sequence was further identified as Gln-Gly-Val-Pro (QGVP) by electrospray mass spectrometry (ESI-MS). QGVP formed 6 hydrogen bonds with the active site of ACE, which is responsible for reducing α-helix structure content of ACE causing subsequent inactivation. Moreover, it showed no significant cytotoxicity toward human umbilical vein endothelial cells (HUVECs), and significantly induced phosphorylation of endothelial nitric oxide synthase (p-eNOS) and decreased endothelin 1 (END1) expression in angiotensin I (Ang I)-treated HUVECs, demonstrating the potential antihypertensive effect. The peptide QGVP hydrolyzed from distilled spent grain proteins of Chinese strong-flavor Baijiu was expected to be used as a food ingredient to prevent or co-treat hypertension with other chemical drugs.

Metabolite-Based Mutualistic Interaction between Two Novel Clostridial Species from Pit Mud Enhances Butyrate and Caproate Production.

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