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.

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.

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.

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.

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 ß-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.

Lentilactobacillus laojiaonis sp. nov., isolated from the mud in a fermentation cellar for the production of Chinese liquor.

A novel Gram-stain-positive, rod-shaped, non-motile bacterial strain, designated IM3328T, was isolated from a mud cellar which has been continuously used over hundreds of years for the fermentative production of Chinese strong-flavour baijiu. It is asporogenous, facultative anaerobic and does not exhibit catalase activity. Strain IM3328T can grow at pH 4.5-8.5 (optimum, pH 7.0), 15-45 °C (optimum, 37 °C), with 0-75% (w/v) ethanol with and 0-6% (w/v) NaCl. The API 50CH assay revealed that strain IM3328T can metabolize l-arabinose, d-ribose, d-xylose, d-glucose, d-fructose, d-mannose, N-acetylglucosamine, gluconate, methyl ß-d-pyranoside, methyl α-d-glucopyranoside, methyl α-d-glucopyranoside and raffinose among the 49 studied carbon sources. Lactic acid, acetic acid, ethanol, isopentanol and butyl acetate are he predominant metabolites in the fermentation broth of strain IM3328T when cultured in liquid de Man, Rogosa and Sharpe medium under micro-aerobic or anaerobic conditions. The polar lipids of strain IM3328T consist of diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid, two unidentified glycolipids and two unidentified lipids. The major cellular fatty acids (≥10%) consist of C16 : 0, C18:1 ω9c and summed feature 7. The cell wall contains ribose, glucose, galactose, lysine, alanine, glutamic acid and aspartic acid. The complete genome of strain IM3328T contains a circular chromosome of 1242019 bp with 1242 genes and 33 mol% G+C content. On the basis of the 16S rRNA gene phylogenetic tree, Lentilactobacillus senioris DSM 24302T (95.9% similarity), Lentilactobacillus rapi DSM 19907T (95.7% similarity) and Lentilactobacillus parabuchneri DSM 5707T (95.1% similarity) were chosen to compare with strain IM3328T to reveal the physiological differences. The low average nucleotide identity values (69.7-71.2%) between strain IM3328T and phylogenetically related reference strains demonstrated that this strain represents a novel species of the genus Lentilactobacillus, and the name Lentilactobacillus laojiaonis sp. nov. (type strain IM3328T=CGMCC 1.18832T=JCM 34630T) is proposed.

Analysis of bacterial diversity and genetic evolution of Lacticaseibacillus paracasei isolates in fermentation pit mud.

AIMS: Since little is known about the genetic diversity of lactic acid bacteria (LAB) isolates from the fermentation pit mud (FPM), we sought to evaluate the bacterial structure, identify the LAB isolates and investigate the genotype and genetic diversity of the LAB isolates. METHODS AND RESULTS: Using high-throughput MiSeq sequencing, we identified seven dominant bacterial genera in FPM. Lactobacillus had the highest abundance. We isolated 55 LAB strains. These isolates were all identified as Lacticaseibacillus paracasei. Using an extant multilocus sequence typing (MLST) scheme, isolates were assigned to 18 sequence types (STs) and three clonal complexes. ST1, the largest group, mainly comprised FPM isolates. Niche-specific ST2 to ST18 only contained FPM isolates. Isolates could be divided into four lineages, with most assigned to Lineage 1. Only one FPM isolate was classified as L. paracasei subsp. paracasei. Other isolates could not be classified at the subspecies level using the seven MLST loci. CONCLUSIONS: Lactobacilli account for a high proportion of bacteria in pit mud. Based on the traditional culture method, L. paracasei was the dominant species, and these isolates exhibit a high ethanol tolerance, high intraspecific diversity and specific genetic profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: The study described the characterization of FPM bacterial diversity, giving an insight into the genetic diversity of L. paracasei strains present in FPM.

Analysis of microbial community diversity and physicochemical factors in pit mud of different ages based on high-throughput sequencing.

In this study, Illumina MiSeq/NovaSeq high-throughput sequencing technology was used to sequence the terminal DNA fragments of microbial communities in Wuliangye pit mud. The results showed that there were 5 dominant bacterial phyla and 13 dominant bacterial genera in the pit mud, which belonged to 4 phyla, mainly Firmicutes. There were 3 dominant fungal phyla and 5 dominant fungal genera in cellar mud, which belonged to 2 phyla and concentrated in Ascomycota. According to the statistical data, the low pH value cellar pool is more conducive to the enrichment of acid-resistant or acid-biased bacteria, which is the key to flavor formation. In addition, the components of ammonium nitrogen, available phosphorus and available potassium in pit mud need to be replenished in time. In addition, sampling time, fermentation time, temperature, and other external environments also have certain effects on microbial diversity and abundance in the pit. With the use of cellars, various types of microorganisms are constantly evolving to adapt to the environment inside the pits. The succession rule of microbe in pit mud was preliminarily revealed, which provided the basis for improving the quality and technical development of Wuliangye.

Adaptability of a Caproate-Producing Bacterium Contributes to Its Dominance in an Anaerobic Fermentation System.

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. IMPORTANCE The Chinese strong aroma-type liquor (Baijiu) fermentation environment is a typical medium-chain fatty acid-producing system with complex nutrients. Although several studies have revealed the correlation between microbial community composition and abiotic factors, the adaptation mechanisms of dominant species to abiotic environment are still unknown in this special anaerobic habitat. This study identified the predominant CPB in Chinese strong aroma-type Baijiu fermentation system. Metabolic versatility and flexibility of the dominant CPB with a small-size genome indicated that this bacterium can effectively exploit available carbon and nitrogen sources, which could be a key factor to promote its ecological success in a multispecies environment. The understanding of growth and metabolic features of the CPB responsible for its dominance in microbial community will not only contribute to the improvement of Chinese strong aroma-type Baijiu production but also expand its potential industrial applications in caproate production.

Mining the Factors Driving the Evolution of the Pit Mud Microbiome under the Impact of Long-Term Production of Strong-Flavor Baijiu.

The mud cellar creates a unique microenvironment for the fermentation of strong-flavor baijiu (SFB). Recent research and long-term practice have highlighted the key roles of microbes inhabiting pit mud in the formation of SFB's characteristic flavor. A positive correlation between the quality of SFB and cellar age was extracted from practice; however, the evolutionary patterns of pit mud microbiome and driving factors remain unclear. Here, based on the variation regularity analysis of microbial community structure and metabolites of samples from cellars of different ages (∼30/100/300 years), we further investigated the effects of lactate and acetate (main microbial metabolites in fermented grains) on modulating the pit mud microbiome. Esters (50.3% to 64.5%) dominated the volatile compounds identified in pit mud, and contents of the four typical acids (lactate, hexanoate, acetate, and butyrate) increased with cellar age. Bacteria (9.5 to 10.4 log10 [lg] copies/g) and archaea (8.3 to 9.1 lg copies/g) mainly constituted pit mud microbiota, respectively dominated by Clostridia (39.7% to 81.2%) and Methanomicrobia (32.8% to 92.9%). An upward trend with cellar age characterized the relative and absolute abundance of the most predominant bacterial and archaeal genera, Caproiciproducens and Methanosarcina. Correlation analysis revealed significantly (P < 0.05) positive relationships between the two genera and major metabolites. Anaerobic fermentation with acetate and lactate as carbon sources enhanced the enrichment of Clostridia, and furthermore, the relative abundance of Caproiciproducens (40.9%) significantly increased after 15-day fed-batch fermentation with lactate compared with the initial pit mud (0.22%). This work presents a directional evolutionary pattern of pit mud microbial consortia and provides an alternative way to accelerate the enrichment of functional microbes. IMPORTANCE The solid-state anaerobic fermentation in a mud cellar is the most typical feature of strong-flavor baijiu (SFB). Metabolites produced by microbes inhabiting pit mud are crucial to create the unique flavor of SFB. Accordingly, craftspeople have always highlighted the importance of the pit mud microbiome and concluded by centuries of practice that the production rate of high-quality baijiu increases with cellar age. To deepen the understanding of the pit mud microbiome, we determined the microbial community and metabolites of different-aged pit mud, inferred the main functional groups, and explored the forces driving the microbial community evolution through metagenomic, metabolomic, and multivariate statistical analyses. The results showed that the microbial consortia of pit mud presented a regular and directional evolutionary pattern under the impact of continuous batch-to-batch brewing activities. This work provides insight into the key roles of the pit mud microbiome in SFB production and supports the production optimization of high-quality pit mud.

Aminipila luticellarii sp. nov., an anaerobic bacterium isolated from the pit mud of strong aromatic Chinese liquor, and emended description of the genus Aminipila.

A novel mesophilic, aerotolerant anaerobic bacterium, designated JN-18T, was isolated from the pit mud of a strong aromatic Chinese liquor. According to a 16S rRNA gene sequence analysis, it had the highest sequence similarity to Aminipila butyrica DSM 103574T (95.69%). The G+C content of its genomic DNA was 43.39 mol%. The cells were Gram-stain-negative, slightly curved rods with flagella. Optimum growth was observed at 37 °C, pH 6.5 and without extra addition of NaCl. Strain JN-18Tutilized amino acids (l-alanine, l-arginine, l-asparagine, l-lysine, l-methionine, l-serine and l-threonine), malate and pyruvate, and used l-arginine and l-lysine to produce acetate, butyrate, H2, and CO2. The major cellular fatty acids of strain JN-18T were C14:0, C16:0 DMA and C18:1 cis-9 DMA. The carbohydrate composition of the cell wall predominantly included galactose, glucose and rhamnose. Based on its phylogenetic, phenotypic, physiological and biochemical characteristics, strain JN-18T was classified as a representative of a novel species within the genus Aminipila, for which the name Aminipila luticellarii sp. nov. is proposed. The type strain is JN-18T (=CCAM 412T=JCM 39126T).

Novisyntrophococcus fermenticellae gen. nov., sp. nov., isolated from an anaerobic fermentation cellar of Chinese strong-flavour baijiu.

A Gram-stain-negative, coccus-shaped, obligately anaerobic, non-motile and non-spore-forming bacterium, designated strain JN500902T, was isolated from the mud in a fermentation cellar used continuously over 30 years for Chinese strong-flavour baijiu production. Colonies were white, circular, convex and smooth-edged. Growth was observed at 20-40 °C (optimum, 37 °C), at pH 5.0-10 (optimum, pH 7.5), with 0-2 % (w/v) NaCl and with 0-4 % (v/v) ethanol. The Biolog assay demonstrated positive reactions of strain JN500902T in the metabolism of l-fucose and pyruvate. The predominant cellular fatty acids (>10 %) consisted of C16 : 0 and C14 : 0. The major end metabolites of strain JN500902T were acetic acid and ethanol when incubated anaerobically in liquid reinforced clostridial medium. Acetate was the major organic acid end product. The complete genome size of strain JN500902T was 3 420 321 bp with 3327 identified genes. The G+C content was 43.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain JN500902T with the family Lachnospiraceae, having low sequence similarity (92.8 %) to the nearest type strain, Syntrophococcus sucromutans DSM 3224T and forming a clearly distinct branch. Core genome phylogenetic analysis of the isolate and 134 strains belonging to the family Lachnospiraceae also revealed that strain JN500902T was well-separated from other genera of this family as a monophyletic clade. The average nucleotide identity and amino acid identity values between strain JN500902T and 134 Lachnospiraceae strains were less than 74 and 65 %, respectively. Considering its polyphasic characteristics, strain JN500902T represents a novel genus and species within the family Lachnospiraceae, for which the name Novisyntrophococcus fermenticellae gen. nov., sp. nov. is proposed. The type strain is JN500902T (=CICC 24502T=JCM 33939T).

Blautia liquoris sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese strong-flavour liquor.

A novel Gram-positive, non-motile, non-flagellated, strictly anaerobic, non-spore-forming and dumbbell-shaped, coccoid- or chain-shaped bacterium, designated strain LZLJ-3T, was isolated from a mud fermentation cellar which has been used for the production of Chinese strong-flavour liquor for over 100 years. Strain LZLJ-3T grew at 20-40 °C (optimum, 37 °C), at pH 6.0-8.0 (optimum, pH 8.0) and with NaCl concentrations up to 1 % (w/v; optimum, 0 %). Phylogenetic trees established based on 16S rRNA gene sequences showed that strain LZLJ-3T belonged to the genus Blautia of the family Lachnospiraceae, with the highest sequence similarity to Blautia stercoris GAM6-1T (91.7 %) and Blautia faecicola KGMB01111T (91.7 %). Comparative genome analysis showed that the orthologous average nucleotide identity (OrthoANI) and genome-to-genome distance (GGD) values between strain LZLJ-3T and B. stercoris GAM6-1T were respectively 69.1 and 22.9 %; the OrthoANI and GGD values between strain LZLJ-3T and B. faecicola KGMB01111T were respectively 70.86 and 36 % . The DNA G+C content of strain LZLJ-3T genome was 42.1 mol%. The predominant celluar fatty acids (>10 %) of strain LZLJ-3T were C16 : 0 FAME (27.9 %), C14 : 0 FAME (17.6 %) and C16 : 0 DMA (13.0 %). Arabinose, glucose and maltose could be utilized by strain LZLJ-3T as sole carbon sources for growth, with weak utilization of raffinose and l-fucose. API ZYM analysis gave positive reactions with α-galactosidase, ß-galactosidase, α-glucosidase and ß-glucosidase. The major end product of glucose fermentation was acetic acid. Based on the results of phenotypic, genotypic and phylogenetic analyses, strain LZLJ-3T is considered to represent a novel species of Blautia, for which the name Blautia liquoris sp. nov. is proposed. The type strain is LZLJ-3T (=KCTC 25163T=CGMCC 1.5299T=JCM 34225T).

Evolving the core microbial community in pit mud based on bioturbation of fortified Daqu.

Directional stress is an effective measure to change the community structure and improve the bioactivity of pit mud (PM). In this study, the addition of fortified Daqu to artificial PM (APM) was intended to disturb the microbial community and further affect metabolites. To evaluate the effect of fortified Daqu on culturing APM, the microbial communities of APM with or without the addition of fortified Daqu were investigated by fluorescence in situ hybridization and Illumina MiSeq. The results indicated that microbes (Clostridium sp., Clostridium kluyveri, hydrogenotrophic methanogens, and acetotrophic methanogens) related to the production of key aroma compounds increased notably when fortified Daqu was added. In particular, the hydrogenotrophic and acetotrophic methanogens increased by 6.19- and 4.63-fold after 30 days of culture. Subsequently, metabolites (organic acids, volatile compounds) were also analyzed by HPLC (high-performance liquid chromatography) and HS-SPME-GC-MS (headspace solid phase microextraction - gas chromatography - mass spectrometry). The results showed that the content of butyric acid and hexanoic acid was significantly higher when fortified Daqu was added to APM. In addition, the proportion of esters and phenols was also higher than in APM without fortified Daqu. A survey of the microbial compositions of APMs with or without added fortified Daqu indicated that the microbial community evolves into a functional community favoring liquor brewing. We have developed a novel process by disturbing the community diversity.

Analysis of the difference between aged and degenerated pit mud microbiome in fermentation cellars for Chinese Luzhou-flavor baijiu by metatranscriptomics.

BACKGROUD: Chinese Luzhou-flavor baijiu (LFB) was fermented in an underground cellar, and the bottom and side of the cellar were covered with pit muds (PMs), where the metabolic activity of the microorganisms had a significant effect on the LFB quality. PMs can be divided into aged pit mud (AP) and degenerated pit mud (DP), thus, the qualities of LFB generated from AP and DP were different. In this essay, metatranscriptomics method was applied to illustrate the differences of the two PMs, as well as to search out the pivotal microorganisms and genes influencing the quality of LFB. RESULTS: Archaea, Clostridium and some thermophilic microorganisms might bring significant effect in AP, while the active eukaryota and Anaeromyxobacter would cause degeneration in PM. Also, the metabolism of carbohydrate and amino acid were more active in AP. What is more, carbohydrate, amino acid and their derivant can produce important organic acids via the activity of the microorganisms in PMs. There were eight critical enzymes noticed in the organic acids metabolic pathway, which were more actively expressed in AP, demonstrating active expression of the critical genes related to organic acid metabolism could have a positive effect on LFB quality. CONCLUSION: This study identified specific differences in active microorganisms, active expressed genes and the expression levels of key genes in vital metabolic pathway between AP and DP. Which may be the actual reason for the differences in the quality of LFB made from different PMs. Mastering these results will provide assistance to improve the quality of LFB. © 2021 Society of Chemical Industry.

Co-occurrence patterns among prokaryotes across an age gradient in pit mud of Chinese strong-flavor liquor.

It is widely believed that the quality and characteristics of Chinese strong-flavor liquor (CSFL) are closely related to the age of the pit mud; CSFL produced from older pit mud tastes better. This study aimed to investigate the alteration and interaction of prokaryotic communities across an age gradient in pit mud. Prokaryotic microbes in different-aged pit mud (1, 6, and 10 years old) were analyzed by Illumina MiSeq sequencing of the 16S rRNA gene. Analysis of the 16S rRNA gene indicated that the prokaryotic community was significantly altered with pit mud age. There was a significant increase in the genera Methanosarcina, Methanobacterium, and Aminobacterium with increased age of pit mud, while the genus Lactobacillus showed a significant decreasing trend. Network analysis demonstrated that both synergetic co-occurrence and niche competition were dominated by 68 prokaryotic genera. These genera formed 10 hubs of co-occurrence patterns, mainly under the phyla Firmicutes, Euryarchaeota, and Bacteroidetes, playing important roles on ecosystem stability of the pit mud. Environmental variables (pH, NH4+, available P, available K, and Ca2+) correlated significantly with prokaryotic community assembly. The interaction of prokaryotic communities in the pit mud ecosystem and the relationship among prokaryotic communities and environmental factors contribute to the higher quality of the pit mud in older fermentation pits.

Profiling the effects of physicochemical indexes on the microbial diversity and its aroma substances in pit mud.

Microbial diversity of pit mud (PM) plays a significant role in Baijiu's flavour. Here we explored the microbial community structures and aroma substances of Wenwang Winery with high-throughput sequencing coupling with headspace solid-phase microextraction-gas chromatography-mass spectrometry. We discovered that the odorant was mainly derived from 14 aroma compounds because of their OAVs ≥ 1 (OAV, the ratio of substance concentration to aroma threshold; s, on behalf of the plural), such as ethyl hexanoate (2438), ethyl octanoate (975), caproic acid (52) and etc. Moreover we also revealed that Lactobacillaceae (97·08%) was the mainly bacterial microbial community in 2-year-old PM, companied by the primarily fungi including Aspergillaceae (55·45%), Unclassified Ascomycota (11·13%) and Dipodascaceae (5·72%). Compared with the 2-year-old PM, bacterial floras in 20-year-old PM and 30-year-old PM were more abundant (i.e. Dysgonomonadaceae, Clostridium and Synerggstaceas), while no fungi were detected. Besides, the physicochemical analysis showed that the content of Lactobacillaceae was inversely associated with moisture, pH and ammonia nitrogen. By further Spearman's correlation coefficient analysis, we verified that the content of Lactobacillaceae was positively correlated with ethyl hexanoate, while negatively correlated with ethyl octanoate and caproic acid. Meanwhile, ethyl octanoate and caproic acid were positively correlated with most flora including Ruminococcaceae, Dysgonomonadaceae and Clostridiacea, which were related to physicochemical indexes. This work demonstrates promise for adjusting the physicochemical indexes of PM to affect the micro-organisms and aroma, which may provide a reference for the production of high-quality Baijiu.

Clostridium fermenticellae sp. nov., isolated from the mud in a fermentation cellar for the production of the Chinese liquor, baijiu.

A novel Gram-stain-positive, rod-shaped, obligately anaerobic, non-motile, spore-forming and binary fission encapsulated bacterium, designated strain JN500901T, was isolated from a mud cellar which has been continuously used for the fermentation of Chinese strong-flavour baijiu for over 100 years. Growth of JN500901Toccurred at pH 4.5-8.0 (optimum, pH 5.0), 20-40 °C (37 °C), 0-2 % (w/v) NaCl and 0-10 % (v/v) ethanol. The Biolog assay revealed that strain JN500901T metabolized d-fructose, l-fucose, isomaltulose and l-rhamnose among the 95 studied carbon sources. p-Cresol was the predominant volatile metabolite in the fermentation broth of strain JN500901T incubated in liquid reinforced clostridial medium under anaerobic conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JN500901T belongs to Clostridiumsensu stricto, and shared the highest sequence similarity to Clostridiumcarboxidivorans DSM 15243T (94.2 %), followed by Clostridiumscatologenes DSM 757T (94.1 %). The dominant cellular fatty acids (>10 %) were C16 : 0 FAME (36.6 %), C19 : 0 cyc 9,10 DMA (19.8 %) and C16 : 1 cis 9 DMA (11.8 %). The complete genome of strain JN500901T contained a circular chromosome of 2.812 Mb with 2611 genes and 31.0 mol% G+C content. Comparative genome analysis of the strain JN500901T, Clostridiumcarboxidivorans DSM 15243T and Clostridiumscatologenes DSM 757T revealed 74.5 and 74.8 % average nucleotide identity, respectively. Based on the phenotypic, biochemical and phylogenetic analyses presented here, strain JN500901T is considered to be a novel species of the genus Clostridiumsensustricto, for which the name Clostridium fermenticellae sp. nov. is proposed. The type strain is JN500901T (=CICC 24501T=JCM 32827T).

Use of microbial indicators combined with environmental factors coupled with metrology tools for discrimination and classification of Luzhou-flavoured pit muds.

AIMS: Pit mud is essential for the quality and yield of Chinese Luzhou-flavoured liquor. A reliable and fast method based on the use of microbial indicators combined with environmental factors coupled with metrology tools is needed to discriminate and classify different maturity levels of Luzhou-flavoured pit muds. METHODS AND RESULTS: Firmicutes, Bacteroidetes, Actinobacteria, Lactobacillus, Bacillus, Methanosarcina, Methanocorpusculum, Methanoculleus and Clostridium kluyveri were microbial indicators in Luzhou-flavoured pit muds. They were detected by real-time quantitative PCR. Environmental factors investigated included moisture content, pH, total acid and ammonia nitrogen. Principal component analysis (PCA) and partial least square-discriminant analysis were employed to explore the structure of the data and construct discrimination and classification models by reduction in the data dimensionality. Pit muds were distinguished and classified as new, trend to-be aged and aged. Moisture content and pH were significantly negatively correlated with new pit mud, while pH, total acid, amino nitrogen, Firmicutes, Bacteroidetes, Actinobacteria, Methanosarcina, Methanoculleus and C. kluyveri were significantly positively correlated with aged pit mud. CONCLUSIONS: Microbial indicators combined with environmental factors coupled to metrology tools can reliably and quickly discriminate and classify different maturity levels of Luzhou-flavoured pit muds. SIGNIFICANCE AND IMPACT OF THE STUDY: Modern techniques and metrology tools verified the correctness of the traditional sensory evaluation used in controlling the quality of pit mud, and will contribute to distinguishing different maturity levels of Chinese Luzhou-flavoured pit muds.

Bacillus vini sp. nov. isolated from alcohol fermentation pit mud.

A novel aerobic, Gram-stain-positive, sporogenous, rod-shaped bacterium, designated LAM0415(T), was isolated from an alcohol fermentation pit mud sample collected from Sichuan Luzhou-flavour liquor enterprise in China. The isolate was found to be able to grow at NaCl concentrations of 0-10 % (w/v) (optimum: 1.0 %), 10-50 °C (optimum: 30-35 °C) and pH 3.0-10.0 (optimum: 7.0-8.0). Phylogenetic analysis of 16S rRNA gene sequences indicated that the new isolate belonged to the genus Bacillus and was closely related to Bacillus sporothermodurans DSM 10599(T) and Bacillus oleronius DSM 9356(T), with 98.4 and 97.2 % sequence similarity, respectively. The DNA-DNA hybridization values between strain LAM0415(T) and the two reference strains were 33.3 ± 1.2 and 42.8 ± 0.8 %, respectively. The genomic DNA G+C content was 35.2 mol% as determined by the T m method. The major fatty acids were determined to be iso-C15:0, anteiso-C15:0 and anteiso-C17:0. The predominant menaquinones were identified as MK7 and MK8. The major polar lipids were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified phospholipid and four unidentified glycolipids. The diagnostic amino acid of the cell wall peptidoglycan was determined to be meso-diaminopimelic acid. On the basis of its phenotypic, phylogenetic and chemotaxonomic characteristics, strain LAM0415(T) (=ACCC 06413(T) = JCM 19841(T)) represents the type strain of a novel species of the genus Bacillus, for which the name Bacillus vini sp. nov. is proposed.