From fungi to lactic acid bacteria: Dominant microorganisms govern stage-specific esters biosynthesis in pit microecology of strong-flavor baijiu.

Targeted microbial collaboration to enhance key flavor metabolites by inoculating Clostridium tyrobutyricum and Saccharomyces cerevisiae in the strong-flavor Baijiu simulated fermentation system

The Chinese alcoholic beverage strong-flavor baijiu (SFB) gets its characteristic flavor during fermentation in cellars lined with pit mud. Microbes in the pit mud produce key precursors of flavor esters. The maturation time of natural pit mud of over 20 years has promoted attempts to produce artificial pit mud (APM) with a shorter maturation time. However, knowledge about the molecular basis of APM microbial dynamics and associated functional variation during SFB brewing is limited, and the role of this variability in high-quality SFB production remains poorly understood. We studied APM maturation in new cellars until the fourth brewing batch using 16S rRNA gene amplicon sequencing, quantitative PCR, metaproteomics, and metabolomics techniques. A total of 36 prokaryotic classes and 195 genera were detected. Bacilli and Clostridia dominated consistently, and the relative abundance of Bacilli decreased along with the APM maturation. Even though both amplicon sequencing and quantitative PCR showed increased abundance of Clostridia, the levels of most of the Clostridium proteins were similar in both the first- and fourth-batch APM samples. Six genera correlated with eight or more major flavor compounds in SFB samples. Functional prediction suggested that the prokaryotic communities in the fourth-batch APM samples were actively engaged in organic acid metabolism, and the detected higher concentrations of proteins and metabolites in the corresponding metabolic pathways supported the prediction. This multi-omics approach captured changes in the abundances of specific microbial species, proteins, and metabolites during APM maturation, which are of great significance for the optimization of APM culture technique.IMPORTANCE Strong-flavor baijiu (SFB) accounts for more than 70% of all Chinese liquor production. In the Chinese baijiu brewing industry, artificial pit mud (APM) has been widely used since the 1960s to construct fermentation cellars for production of high-quality SFB. To gain insights at the systems level into the mechanisms driving APM prokaryotic taxonomic and functional dynamics and into how this variation is connected with high-quality SFB production, we performed the first combined metagenomic, metaproteomic, and metabolomic analyses of this brewing microecosystem. Together, the multi-omics approach enabled us to develop a more complete picture of the changing metabolic processes occurring in APM microbial communities during high-quality SFB production, which will be helpful for further optimization of APM culture technique and improvement of SFB quality.

ABSTRACTThe 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.

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.

Insecticide resistance in the aphid Myzus persicae (Sulzer): chromosome location and epigenetic effects on esterase gene expression in clonal lineages

Comparative analysis of the differences among Langya flavor Baijiu and strong and soy sauce flavor Baijiu by targeted flavor analysis

Potential of ethylene in alleviating cold-induced volatile esters loss of ‘Nanguo’ pears by regulating the lipoxygenase pathway

Effects of biofilm and co-culture with Bacillus velezensis on the synthesis of esters in the strong flavor Baijiu

Optimizing Baijiu fermentation with high-yield ethyl caproate-producing Candida parapsilosis strain

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

BackgroundAroma is an important organoleptic quality for fruit and has a large influence on consumer preference. Kiwifruit esters undergo rapid and substantial changes contributing to the flavor during fruit ripening. Part of enzymes and their coding genes have been indicated potential candidates for flavor-related esters synthesis. However, there still exist obvious gaps in the biosynthetic pathways of esters and the mechanisms regulating ester biosynthesis in kiwifruit remain unknown.ResultsUsing gas chromatography-mass spectrometry (GC-MS), volatile compounds of kiwifruit were quantified in response to ethylene (ETH, 100 μl/l, 24 h, 20 °C) and 1-methylcyclopropene (1-MCP, 1 μl/l, 24 h, 20 °C). The results indicated that esters showed the most substantial changes enhanced by ethylene and were inhibited by 1-MCP. Correlations between RNA-seq results and concentrations of esters, constructed using Weighted Gene Co-Expression Network Analysis (WGCNA) indicated that three structural genes (fatty acid desaturase, AdFAD1; aldehyde dehydrogenase, AdALDH2; alcohol acyltransferase, AdAT17) had similar expression patterns that paralled the changes in total ester content, and AdFAD1 transcripts exhibited the highest correlation. In order to search for potential regulators for ester biosynthesis, 14 previously reported ethylene-responsive transcription factors (TFs) were included in the correlation analysis with esters and their biosynthetic genes. Using dual-luciferase assay, the in vivo regulatory activities of TFs on ester biosynthetic gene promoters were investigated and the results indicated that AdNAC5 and AdDof4 (DNA binding with one finger) trans-activated and trans-suppressed the AdFAD1 promoter.ConclusionsThe present study advanced the molecular basis of ripening-related ester biosynthesis in kiwifruit by identifying three biosynthetic related genes AdFAD1, AdALDH2 and AdAT17 by transcriptome analysis, and highlighted the function of two TFs by transactivation studies.

Strong-flavor baijiu is a traditional distilled alcoholic beverage with a long history in China. The fermented grains play a pivotal role in the production of baijiu. The purpose of this study was to evaluate and compare the microbiota and flavor substances present in fermented zaopei (ZP) from pits of different ages. High-throughput sequencing, headspace solid-phase microextraction gas chromatography-mass spectrometry, principal component analysis, community composition analysis, and redundancy analysis were used to analyze and evaluate the impact of environmental factors on microbial communities and flavor substances. Six genera of bacteria (e.g., Caproiciproducens, Syntrophaceticus, Sedimentibacter, Hydrogenispora, Pelotomaculum and Bacillus) and seven genera of fungi (Cladosporium, Debaryomyces, Dipodascus, Auxarthron, Cephalotrichum, unclassified Stachybotryaceae, unclassified Microascaceae and Cephalotrichum) notably affected the production of hexanoic acid (an important flavor compound). Moisture and alcohol content also had considerable effects on the production of the flavor compounds such as ethyl lactate, hexanoic acid, and ethyl hexanoate. The profiles of volatile compounds present in ZP were different between the aged and new pits; these profiles were mainly reflected in the concentration and types of alcohols, aldehydes, esters, and aromatic compounds. This paper provides a comprehensive overview of the physicochemical parameters, flavor substances, and microbial population distribution of ZP. Characterization of various ZP samples help to elucidate the fermentation mechanisms and offer a theoretical reference to control and enhance the quality of Baijiu. © 2021 Society of Chemical Industry.

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

Characterization of the differences in aroma-active compounds in strong-flavor Baijiu induced by bioaugmented Daqu using metabolomics and sensomics approaches.

Arecae semen, which is derived from the dried ripe seed of Areca catechu L., has been commonly used as one of the major traditional Chinese medicines (TCMs). Three types of crude herbal preparations, namely, raw Arecae semen (AS), Arecae semen tostum (SAS), and Arecae semen carbonisata (FAS), are available for different clinical applications in TCMs. Although aflatoxin contamination in Arecae semen has been reported preliminarily, only a few studies have been conducted on fungal contamination. In this study, the presence of fungi on the surface of three Arecae semen (AS, SAS, and FAS) that collected from four provinces were investigated using high-throughput sequencing and internal transcribed spacer 2. Results showed that the phyla Ascomycota (75.45%) and Basidiomycota (14.29%) and the genera Wallemia (7.56%), Botryosphaeria (6.91%), Davidiella (5.14%), and Symbiotaphrina (4.87%) were the dominant fungi, and they presented significant differences in four areas and three processed products (p < 0.05). The α-diversity and network complexity exhibited significant differences in the four sampling locations (p < 0.05), with higher in Yunnan (Chao 1, 213.45; Shannon, 4.61; average degree, 19.96) and Hainan (Chao 1, 198.27; Shannon, 4.21; average degree, 22.46) provinces. Significant differences were noted in the three processed samples; and SAS group had highest α-diversity (Chao 1, 167.80; Shannon, 4.54) and network complexity (average degree, 18.32). In conclusion, the diversity and composition of microbiome on the surface of Arecae semen were shaped by sampling location and processing methods. This work provides details on the surface microbiome of Arecae semen samples and highlights the importance of roles of origin and processing methods in microbiomes, ensuring drug efficacy and food safety.