Chinese Liquor Production Research Articles

Exploring the contribution of temperature-adapted microbiota to enzyme profile of saccharification in Daqu using metagenomics and metaproteomics

Temperature is a critical factor that influences the microbiota in Daqu, which provides essential enzymes for the saccharification process in Baijiu (Chinese liquor) production. However, the relationship between temperature-adapted microbiota and saccharification properties of Daqu remains unclear. In this study, a profile of 23 major saccharifying enzymes and 40 responsible genera was obtained from the metagenomic data of medium- and high-temperature Daqu (MT- and HT-Daqu). The high-temperature pattern (65 °C) primarily affected the saccharification potential of Daqu by promoting Bacillales (fold-change: 0.25–0.75), whereas inhibiting Lactobacillales (fold-change: 3.80–5.60) and Mucorales (fold-change: 5.65–7.09). Eurotiales only exhibited a slight increase (fold-change: 1.01–1.24) to low-temperature pattern (56 °C). Metaproteomic analysis further revealed a notable contribution of Eurotiales in facilitating saccharification. Amylolytic enzymes were primarily derived from Eurotiales species (Byssochlamys spectabilis, Monascus purpureus, Rasamsonia emersonii, and Thermomyces lanuginosus) and one Mucorales species, Lichtheimia romasa. Cellulolytic enzymes were produced by Thermoascus aurantiacus and Paecilomyces sp. Among them, L. romasa was the principal contributor under the low-temperature pattern. Whereas, in HT-Daqu, B. spectabilis, M. purpureus, and T. lanuginosus were the major contributors, as L. romasa is susceptible to higher temperatures. This study offers valuable guidance for targeted optimization of industrial production.

Demography and fitness of Sesamia inferens Walker (Lepidoptera: Noctuidae) on three important gramineous crops

BackgroundPink stem borer, Sesamia inferens (Walker), a major rice pest, has rarely been reported to cause serious damage to sorghum in China. During a survey of insect pests in glutinous sorghum for Chinese liquor production areas in Guizhou Province, it was found that S. inferens has caused serious damage to local sorghum in recent years.MethodsIn this context, we assessed the adaptation of S. inferens to three gramineous crop host plants (maize, rice and sorghum) based on age-stage, two-sex life table theory.ResultsSesamia inferens had the highest development time, survival rate and total longevity on rice, followed by on sorghum and maize. There was no significant difference in intrinsic rate of increase and finite rate of increase among maize (r = 0.0702 day−1, λ = 1.0727 day−1), sorghum (r = 0.0681 day−1, λ = 1.0705 day−1) and rice (r = 0.0645 day−1, λ = 1.0666 day−1), but the mean generation period (T) was significantly higher for populations reared on rice (53.84 days) than on sorghum (49.53 days) and maize (47.31 days).ConclusionsThe results of our study indicate that S. inferens was able to complete the full developmental cycle on all three host plants. This study further supports that glutinous sorghum is an adaptable host plant for S. inferens.

Comprehensive analysis of the relationship between peptide profiles and microbial diversity in the Chinese Jiang‐Flavour Daqu

SummaryJiang‐Flavour Daqu (JFDQ) is a type of grain‐based fermentation starter used in Chinese liquor production. This study aimed to monitor the practical production of JFDQ and improve brewing techniques by analysing the microbial diversity and peptide profiles in JFDQ with different colours (WDQ, YDQ and BDQ) using 16S rRNA gene sequencing and peptidomics technology. A total of 110 operational taxonomic units (OTUs) were identified, with BDQ containing the most (98). Bacillus was predominant in WDQ (77.5%) and YDQ (82.3%) but decreased sharply in BDQ (29.0%). Most peptides were identified in WDQ (406), followed by YDQ (282) and BDQ (90). The content of peptides increased (7.16–9.13 g/100 g) from WDQ, YDQ to BDQ, whereas the number of peptides decreased (406–90). This phenomenon may result from a synergistic effect of the microbes in generating and consuming peptides as exogenous nitrogen sources. Additionally, six novel antimicrobial peptides were found against Escherichia coli and Staphylococcus aureus, indicating an antagonistic relationship between antimicrobial peptides and microbes in JFDQ. This study provides a credible direction for investigating the interaction between microbes and peptides in JFDQ, which could guide the practical production more scientifically.

Metaproteomics profiling of the microbial communities in fermentation starters (Daqu) during multi-round production of Chinese liquor.

The special flavor and fragrance of Chinese liquor are closely related to microorganisms in the fermentation starter Daqu. The changes of microbial community can affect the stability of liquor yield and quality. In this study, we used data-independent acquisition mass spectrometry (DIA-MS) for cohort study of the microbial communities of a total of 42 Daqu samples in six production cycles at different times of a year. The DIA MS data were searched against a protein database constructed by metagenomic sequencing. The microbial composition and its changes across production cycles were revealed. Functional analysis of the differential proteins was carried out and the metabolic pathways related to the differential proteins were explored. These metabolic pathways were related to the saccharification process in liquor fermentation and the synthesis of secondary metabolites to form the unique flavor and aroma in the Chinese liquor. We expect that the metaproteome profiling of Daqu from different production cycles will serve as a guide for the control of fermentation process of Chinese liquor in the future.

Quantitative metaproteomics reveals composition and metabolism characteristics of microbial communities in Chinese liquor fermentation starters.

Daqu, the Chinese liquor fermentation starter, contains complex microbial communities that are important for the yield, quality, and unique flavor of produced liquor. However, the composition and metabolism of microbial communities in the different types of high-temperature Daqu (i.e., white, yellow, and black Daqu) have not been well understood. Herein, we used quantitative metaproteomics based on data-independent acquisition (DIA) mass spectrometry to analyze a total of 90 samples of white, yellow, and black Daqu collected in spring, summer, and autumn, revealing the taxonomic and metabolic profiles of different types of Daqu across seasons. Taxonomic composition differences were explored across types of Daqu and seasons, where the under-fermented white Daqu showed the higher microbial diversity and seasonal stability. It was demonstrated that yellow Daqu had higher abundance of saccharifying enzymes for raw material degradation. In addition, considerable seasonal variation of microbial protein abundance was discovered in the over-fermented black Daqu, suggesting elevated carbohydrate and amino acid metabolism in autumn black Daqu. We expect that this study will facilitate the understanding of the key microbes and their metabolism in the traditional fermentation process of Chinese liquor production.

Comparison of the microbial communities in pits with different sealing methods for Chinese strong-flavor liquor production

Chinese liquor fermentation is a typical solid-state fermentation process driven by various natural microflora, which is susceptible to ambient influence. A new-type sealing method using stainless steel as the pit cover was adopted as an alternative solution to mitigate the influence. In this study, we investigated the microbial consortia and the flavor metabolites in both the new-type sealing pit (NSP) and the traditional sealing pit (TSP). The study found the concentrations of most flavor metabolites (39 out of 58) in NSP were higher than TSP, and most of them concentrated at the bottom of both pits. The comparison revealed the diversities of microbes in different layers of pits were different (p < 0.05). The compositions of microbiota in different pits were different, albeit Lactobacillus was the absolute dominant genus in both pits. There were 13 dominant genera in NSP and seven in TSP. Therefore, more correlations between microbes were found in NSP, in which the microbial community was more stable. The interplay between different microbes and metabolites consequently resulted in different association patterns in different pits. These findings elaborate on the reasons behind the preference of the new-type sealing method and are instrumental to the optimization of the fermentation process.

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 β-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.

Comparative Genomics Unveils the Habitat Adaptation and Metabolic Profiles of Clostridium in an Artificial Ecosystem for Liquor Production.

ABSTRACTClostridium inhabiting pit mud (PM) is one of the important bacterial populations for synthesizing flavor compounds of Chinese strong-flavor baijiu. The long-term cereal fermentation with sorghum as the main raw material creates an environment rich in starch, ethanol, and organic acids (mainly lactic acid). However, the genetic factors underpinning Clostridium’s adaptation to PM remain poorly understood. Here, we performed comparative genomic analysis between 30 pit mud-associated (PMA) and 100 non-pit mud-associated (NPMA) Clostridium strains. Comparison analysis of the enrichment of KEGG pathways between PMA and NPMA Clostridium strains showed two-component system, flagellar assembly, and bacterial chemotaxis pathways related to environmental adaptation were enriched in PMA strains. The number of genes encoding alcohol dehydrogenase and l-lactate dehydrogenase in PMA Clostridium strains was significantly higher than that in NPMA, which is helpful for them to adapt to the ethanol- and lactic acid-rich environment. The analysis of carbohydrate-active enzymes demonstrated that glycoside hydrolases (GHs) was the most abundant family in all Clostridium strains, and genes encoding GH4 and GH13, involved in starch and sucrose metabolism, were enriched in PMA Clostridium. Horizontal gene transfer analysis revealed that multiple genes encoding the enzymes involved in carbohydrate and amino acid metabolism were transferred from Bacillus to Clostridium in pit mud. Most of the PMA Clostridium strains had good potential for butyric acid synthesis from ethanol, lactic acid, and starch. Collectively, this study furthers our understanding of the habitat adaptation and metabolic potential of PMA Clostridium strains.IMPORTANCE Pit mud is a typical artificial ecosystem for Chinese liquor production. Clostridium inhabiting pit mud plays essential roles in the flavor formation of strong-flavor baijiu. The relative abundance of Clostridium increased with pit mud quality, further influencing the quality of baijiu. So far, the ecological adaptation of Clostridium to a pit mud-associated lifestyle is largely unknown. Here, comparative genomic analysis of pit mud-associated (PMA) and non-pit mud-associated (NPMA) Clostridium strains was performed. We found genes related to the metabolism of starch, ethanol, and lactic acid were enriched in PMA Clostridium strains, which facilitated their adaptation to the unique brewing environment. In addition, horizontal gene transfer contributed to the adaptation of Clostridium to pit mud. Our findings provide genetic insights on PMA Clostridium strains’ ecological adaptation and metabolic characteristics.

Caproicibacterium lactatifermentans sp. nov., isolated from pit clay used for the production of Chinese strong aroma-type liquor

Two recently reported bacterial strains that were identified as the dominant caproate-producing bacteria in pit clay, were further characterized to determine their phylogeny and taxonomy. The two strains, designated as LBM19010T and JNU-WLY1368, were short rod-shaped, Gram-stain-positive, non-motile and strictly anaerobic. Analysis of the 16S rRNA gene sequences revealed that strains LBM19010T and JNU-WLY1368 shared a 16S rRNA gene sequence similarity of 99.93 % and belonged to a recent proposed genus Caproicibacterium in the family Oscillospiraceae. The proposed type strain, LBM19010T, showed the highest 16S rRNA gene sequence similarity to Caproicibacterium amylolyticum LBM18003T (96.34%), followed by Caproiciproducens galactitolivorans JCM 30532T (94.14 %). The pairwise average nucleotide identity and average amino acid identity values between strains LBM19010T and LBM18003T were 74.84 and 76.18 %, respectively. Growth of strain LBM19010T occurred at pH 4.5-7.5 (optimum, pH 5.0-5.5), 20-40 °C (optimum, 35 °C) and with 0-1 % (w/v) NaCl (optimum, 0 %). Strains LBM19010T and JNU-WLY1368 were both able to ferment several hexoses, disaccharides, starch and lactate but not pentoses. Caproate and butyrate were the major end-products from glucose. The predominant cellular fatty acids (>10 %) of strain LBM19010T were C16 : 0 (56.3 %), C14 : 0 DMA (19.5 %) and C14 : 0 (14.9 %). The identified polar lipids of strain LBM19010T were diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids and nine unidentified glycolipids. Based on phylogenetic, phenotypic and chemotaxonomic evidence, strains LBM19010T and JNU-WLY1368 belong to a novel species of the genus Caproicibacterium, for which the name Caproicibacterium lactatifermentans sp. nov. is proposed. The type strain is LBM19010T (=GDMCC 1.1627T=JCM 33782T).

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, β-galactosidase, α-glucosidase and β-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).

Silage Fermentation on Sweet Sorghum Whole Plant for Fen-Flavor Baijiu.

The technology for producing bioethanol from sweet sorghum stalks by solid-state fermentation has developed rapidly in recent years, and has many similarities with traditional Chinese liquor production. However, the product from sweet sorghum stalks was lacking in volatile flavors, and the level of harmful contents were uncertain, therefore it could not be sold as liquor. In this study, the protein, fat, and tannin in the clusters and leaves of sweet sorghum were utilized to increase the content of flavor compounds in the ethanol product through the anaerobic fermentation of Saccharomyces cerevisiae. Meanwhile, the silage fermentation method was used to extend the preservation time of the raw materials and to further enhance the flavors of Fen-flavor liquor, with ethyl acetate as the characteristic flavor. The effects of different feedstock groups on ethyl acetate, ethyl lactate, methanol, acetaldehyde, acetal, fusel oil, total acid, and total ester were evaluated by analyzing the chemical composition of different parts of sweet sorghum and determined by gas chromatograph. The effect of different fermentation periods on the volatile flavor of sweet sorghum Baijiu was evaluated. The yield of the characteristic volatile flavor was increased by the extension of the fermentation time. Sweet sorghum Baijiu with a high ester content can be used as a flavoring liquor, blended with liquor with a shorter fermentation period to prepare the finished Fen-flavor Baijiu, conforming to the Chinese national standard for sale.

Metagenome and analysis of metabolic potential of the microbial community in pit mud used for Chinese strong-flavor liquor production

Complex microbiomes of pit mud (PM) play significant roles in imbuing flavors and qualities of Chinese strong-flavor liquor (CSFL) during fermentation. However, understanding both of the taxonomic and functional diversity of the whole microorganisms in PM still remain a major challenge. Here, PM microbiomes were investigated based on metagenomic sequencing, assembly and binning. Metagenomic data revealed that Euryarchaeota was the predominant phylum, followed by Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. For further functional exploration, 703 metagenome-assembled genomes (MAGs), including 304 novel strains, 197 novel species, and 94 novel genera were reconstructed. Three primary groups of Firmicutes (n = 406), Euryarchaeota (n = 130) and Bacteroidetes (n = 74), particularly genus of them Syntrophomonas, Thermacetogenium and Clostridium, methanogens (Methanobacterium, Methanoculleus, and Methanosarcina), Proteiniphilum and Prevotella, contained most of metabolic potential genes. Additionally, Chloroflexi was firstly reported to have potential to be involved in the caproic acid (CA) production. Bacteroidetes could be the key phylum to synthesize terpenes, and Armatimonadetes, Firmicutes, Ignavibacteriae and Verrucomicrobia may possess the same metabolic potential as well. Overall, this study will significantly improve our understanding of the diverse PM microbiome and help guide the future exploration of microbial resources for modifying PM fermentation processes.

Identification of water-soluble peptides in distilled spent grain and its angiotensin converting enzyme (ACE) inhibitory activity based on UPLC-Q-TOF-MS and proteomics analysis

Distilled spent grain (DSG) is the biggest by-product in baijiu (Chinese liquor) production, releasing approximately 23.44 million tons every year. Aiming at comprehensive identification of more bioactive peptides, in this work, the new bioassay-guided proteomics and Biolynx peptide sequencer based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) were developed. Moreover, 22 peptides with angiotensin converting enzyme (ACE) inhibitory activities were identified. Seven peptides were successfully quantified using electrospray ionization with triple-quadrupole mass spectrometry (ESI-QQQ-MS) in the multiple reaction monitoring (MRM). Of these identified peptides, Pro-Arg was the most abundant (92.14 μg g−1 dry weight (DW)) and acted as a competitive inhibitor of ACE by molecular docking. Therefore, peptides from DSG can be considered as promising candidates for ACE inhibition; in addition, the new strategy for peptide sequencing can be extended to any food matrices containing peptide mixture or protein hydrolysate.

Industrial applications of selected JFS articles

Industrial applications of selected <i>JFS</i> articles

Basic flavor types and component characteristics of Chinese traditional liquors: A review.

Chinese traditional liquor, a major type of global distilled spirits, offers a unique flavor system acquired across thousands of years of development. Owing to the various raw brewing materials, types of koji, fermentation vessels, and processes used during liquor production, significant differences can occur in the content of flavor chemical components, such as esters, alcohols, aromatics, ketones, nitrogen compounds, acids, and aldehydes in the resulting liquor. Therefore, the liquor can be characterized on the basis of four basic flavors: sauce-, strong-, light-, and rice-aroma, and eight derivative flavors: feng-, sesame-, chi-, te-, mixed-, laobaigan-, herbal-, and fuyu-aroma. In this review, we describe the production and development process of Chinese traditional liquor in detail; summarize the flavor types, flavor chemical composition characteristics, and research progress related to this liquor; and discuss the influence of trace chemical components on liquor flavor, with the aim of laying a theoretical foundation for stabilizing the quality and increasing the yield of traditional liquor.

Detection of Viable and Total Bacterial Community in the Pit Mud of Chinese Strong-Flavor Liquor Using Propidium Monoazide Combined With Quantitative PCR and 16S rRNA Gene Sequencing.

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

Modeling of industrial-scale anaerobic solid-state fermentation for Chinese liquor production

Traditional solid-state fermentation processes can give fluctuating product quality and quantity due to difficulties in control and scale up. This paper describes an engineering study of an industrial-scale anaerobic solid-state fermentation process for Chinese liquor (Baijiu) production, aimed at better understanding of the traditional process, as an initial step for future optimization. This mixed-culture fermentation is done in 0.44-m3 vessels embedded in the soil. At this scale, the fermentation is limited by product inhibition. We developed mathematical models based on the Han-Levenspiel equation for product inhibition, with parameters derived from measured data. The models accurately predicted the concentrations of starch and dry matter. A model with radial conduction into a small soil volume around the fermenter and consecutive vertical conduction into the underlying soil accurately predicted the pit temperature in the heating and cooling phases. This model is very sensitive to the values used for the enthalpies of combustion, meaning that direct measurement of the heat production rate would be preferable. In the industry practice, the fermenter volume can be from around 0.20 to 15.00 m3. The model predicts that overheating will occur not only in larger fermenters, but also in the 0.44-m3 fermenters when the soil temperature is high in summer. Our model predictions are consistent with observed behavior in the industry. Our findings can be used to improve this traditional process, as well as similar systems.

Synergistic Effect of Multiple Saccharifying Enzymes on Alcoholic Fermentation for Chinese Baijiu Production.

Chinese Jiuqu (fermentation starter) provides saccharifying enzymes for baijiu (Chinese liquor) fermentation, which undergoes a simultaneous saccharification and fermentation process. However, the key saccharifying enzymes associated with alcoholic fermentation from Jiuqu and their effects on ethanol production remain poorly understood. In this study, we identified 51 carbohydrate hydrolases in baijiu fermentation by metaproteomics analysis. Through source-tracking analysis, approximately 80% of carbohydrate hydrolases in the baijiu fermentation were provided by Jiuqu Among these enzymes, alpha-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3), from Aspergillus, Rhizomucor, and Rhizopus, were positively related to starch hydrolysis and ethanol production, indicating that they were the key saccharifying enzymes associated with alcoholic fermentation in the baijiu fermentation. Moreover, a combined mixture of alpha-amylase and glucoamylase (in a ratio of 1:6, wt/wt) enhanced ethanol production in a simulative baijiu fermentation under laboratory conditions. This result revealed a synergistic effect of multiple saccharifying enzymes on ethanol production in baijiu fermentation. Our study provides a potential approach to improve the efficiency of saccharification and alcoholic fermentation by optimizing the profile of saccharifying enzymes for fermentation of baijiu and other beverages.IMPORTANCEJiuqu starter provides enzymes to the simultaneous saccharification and fermentation process of baijiu (Chinese liquor) production; however, the key saccharifying enzymes associated with alcoholic fermentation from Jiuqu and their effects on ethanol production remain unclear. We confirmed that Jiuqu was the main source of carbohydrate hydrolases for baijiu fermentation and identified two types of saccharifying enzymes from multiple microbes as the key enzymes associated with alcoholic fermentation. Moreover, a proper combination of multiple saccharifying enzymes could enhance ethanol production in baijiu fermentation. This combination provides an approach to optimize the profile of saccharifying enzymes for enhancing ethanol production in baijiu and other food fermentations.

Structural and Functional Changes in Prokaryotic Communities in Artificial Pit Mud during Chinese Baijiu Production.

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.

Regional aroma characteristics of sorghum for Chinese liquor production

Regional aroma characteristics of sorghum for Chinese liquor production