[Metagenomics unveils the differences in the functions of microbial community of medium-temperature Daqu before and after maturation].

Daqu is the saccharifying, fermenting, and aroma-producing agent used in Baijiu brewing, and its maturation is crucial for obtaining high-quality Daqu. Previous studies have explored the microbial community composition and diversity before and after maturation. However, little is known about the changes in the functions of microbial community. In this study, based on the analyses of enzyme activities and volatile compounds of medium-temperature Daqu before and after maturation, metagenomics was used to analyze the differences in the composition of microbial community and the potential functions, with the aim to explore the microorganisms involved in changes in enzyme activities and important volatiles. The results showed that the moisture (P≤0.05), starch content, liquefying activity, saccharifying activity (P≤0.05), and fermentative activity decreased, while the acidity and esterifying activity (P≤0.05) increased after Daqu maturation. In the meantime, the composition of volatile compounds changed significantly (P=0.001), with significant decreases in the contents of aromatic alcohols and esters as well as significant increases in the contents of pyrazines, ketones, and higher fatty alcohols. The relative abundances of Mucorales (34.8%-23.0%) and Eurotiales (34.3%-20.1%) decreased in matured Daqu, and functional predictions showed these changes decreased the gene abundances of α-amylase, α-glucosidase, alcohol dehydrogenase, and alcohol dehydrogenase (NADP+) (P > 0.05), resulting in lower levels of liquefying activity (P > 0.05), saccharifying activity (P≤0.05), fermentative activity (P > 0.05), as well as aromatic alcohols such as phenylethyl alcohol (P≤0.05). In addition, higher relative abundances of Saccharomycetales (2.9%-16.6%), Lactobacillales (14.9%-23.6%), and Bacillales (0.8%-3.8%) were observed after maturation, and they were conducive to improving the gene abundances of alcohol O-acetyltransferase, carboxylesterase, acetolactate decarboxylase, (R)-acetoin dehydrogenase, and (S)-acetoin dehydrogenase (P≤0.05), resulting in significantly higher levels of esterifying activity and pyrazines (P≤0.05). The microorganisms involved in the changes in enzyme activities and important volatiles before and after Daqu maturation were studied at the gene level in this work, which may facilitate further rational regulation for Daqu production.

Rapid detection of six Oceanobacillus species in Daqu starter using single-cell Raman spectroscopy combined with machine learning.

Many traditional fermented foods and beverages industries around the world request the addition of multi-species starter cultures. However, the microbial community in starter cultures is subject to fluctuations due to their exposure to an open environment during fermentation. A rapid detection approach to identify the microbial composition of starter culture is essential to ensure the quality of the final products. Here, we applied single-cell Raman spectroscopy (SCRS) combined with machine learning to monitor Oceanobacillus species in Daqu starter, which plays crucial roles in the process of Chinese baijiu. First, a total of six Oceanobacillus species (O. caeni, O. kimchii, O. iheyensis, O. sojae, O. oncorhynchi subsp. Oncorhynchi and O. profundus) were detected in 44 Daqu samples by amplicon sequencing and isolated by pure culture. Then, we created a reference database of these Oceanobacillus strains which correlated their taxonomic data and single-cell Raman spectra (SCRS). Based on the SCRS dataset, five machine-learning algorithms were used to classify Oceanobacillus strains, among which support vector machine (SVM) showed the highest rate of accuracy. For validation of SVM-based model, we employed a synthetic microbial community composed of varying proportions of Oceanobacillus species and demonstrated a remarkable accuracy, with a mean error was less than 1% between the predicted result and the expected value. The relative abundance of six different Oceanobacillus species during Daqu fermentation was predicted within 60 min using this method, and the reliability of the method was proved by correlating the Raman spectrum with the amplicon sequencing profiles by partial least squares regression. Our study provides a rapid, non-destructive and label-free approach for rapid identification of Oceanobacillus species in Daqu starter culture, contributing to real-time monitoring of fermentation process and ensuring high-quality products.

Potential mechanism of Qinggong Shoutao pill alleviating age-associated memory decline based on integration strategy.

CONTEXT: Qinggong Shoutao Wan (QGSTW) is a pill used as a traditional medicine to treat age-associated memory decline (AAMI). However, its potential mechanisms are unclear. OBJECTIVE: This study elucidates the possible mechanisms of QGSTW in treating AAMI. MATERIALS AND METHODS: Network pharmacology and molecular docking approaches were utilized to identify the potential pathway by which QGSTW alleviates AAMI. C57BL/6J mice were divided randomly into control, model, and QGSTW groups. A mouse model of AAMI was established by d-galactose, and the pathways that QGSTW acts on to ameliorate AAMI were determined by ELISA, immunofluorescence staining and Western blotting after treatment with d-gal (100 mg/kg) and QGSTW (20 mL/kg) for 12 weeks. RESULTS: Network pharmacology demonstrated that the targets of the active components were significantly enriched in the cAMP signaling pathway. AKT1, FOS, GRIN2B, and GRIN1 were the core target proteins. QGSTW treatment increased the discrimination index from -16.92 ± 7.06 to 23.88 ± 15.94% in the novel location test and from -19.54 ± 5.71 to 17.55 ± 6.73% in the novel object recognition test. ELISA showed that QGSTW could increase the levels of cAMP. Western blot analysis revealed that QGSTW could upregulate the expression of PKA, CREB, c-Fos, GluN1, GluA1, CaMKII-α, and SYN. Immunostaining revealed that the expression of SYN was decreased in the CA1 and DG. DISCUSSION AND CONCLUSIONS: This study not only provides new insights into the mechanism of QGSTW in the treatment of AAMI but also provides important information and new research ideas for the discovery of traditional Chinese medicine compounds that can treat AAMI.

Solibacillus daqui sp. nov., isolated from high-temperature Daqu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS111008T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu, and was characterized by polyphasic taxonomy. This novel isolate grew in the presence of 0-5 % (w/v) NaCl, at pH 6.0-9.0 and 25-45 °C; optimum growth was observed with 1 % (w/v) NaCl, at pH 8.0 and 30 °C. A comparative analysis of the 16S rRNA gene sequence (1461 bp) of strain ZS111008T showed highest similarity to Solibacillus silvestris DSM12223T (96.7%), followed by Solibacillus cecembensis PN5T (96.6%) and Solibacillus isronensis AMCK01000046 (96.5%). The DNA G+C content of strain ZS111008T was 37.21 mol%. The respiratory quinone was identified as menaquinone-7 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and one unknown phospholipid. Lys was detected as the diagnostic diamino acid in the cell wall. Based on morphological characteristics, chemotaxonomic characteristics and physiological properties, strain ZS111008T represents a novel species of the genus Solibacillus, for which the name Solibacillus daqui sp. nov. is proposed. The type strain for this proposed species is ZS111008T (=CGMCC 1.19455T=JCM 35214T).

Preparation of High-Ductility Cement-Calcined Coal-Gangue-Powder-Composite-Based Rapid Repair Material.

Coal gangue is a kind of solid waste. A high-ductility cement-calcined coal-gangue-powder-composite-based rapid repair material (HD-RRM) was prepared by partially replacing cement with calcined coal gangue powder (CCGP) for achieving high ductility and rapid hardening and conforming to the strength requirements of pavement layers. First, the physical and chemical properties and the reactivity of the CCGP were investigated. Second, HD-RRM material was prepared, and its tensile performance characteristic parameters were investigated. Lastly, the hydration products and microstructure of HD-RRM were characterized through tests (e.g., non-evaporable water content, scanning electron microscopy (SEM), X-ray diffraction (XRD), and comprehensive thermogravimetric analysis and differential scanning calorimetry (TG-DSC)). As indicated by the experimental results, the CCGP with a particle size of 1250 mesh exhibited the maximum potential reactivity. The optimal mixing ratio for HD-RRM in the experiments comprised a water-cement ratio of 0.27, a sand-cement ratio of 0.3, a fiber volume fraction of 2%, a cement content of 70%, a CCGP content of 20%, a fly ash (FA) content of 10%, and a superplasticizer content of 0.1%. Using the abovementioned mix design, the prepared HD-RRM was endowed with a 6 h ultimate elongation of 2.75%, an ultimate tensile strength of 7.58 MPa, a compressive strength of 45.4 MPa, and an average crack width of 125.53 µm, which meets the requirements of repair materials and provides a design method for CCGP resource utilization and asphalt concrete road and bridge deck repair.

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

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

Lentibacillus daqui sp. nov., isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterial strain, designated ZS110521T, was isolated from high-temperature Daqu, a starter for production of Chinese Jiang-flavour Baijiu and was characterised by polyphasic taxonomy. This novel isolate grew in the presence of 0-20 % (w/v) NaCl, at pH 6.0-9.0 and 20-50 °C; optimum growth was observed with 8-10 % (w/v) NaCl, at pH 7.0 and 37 °C. A comparative analysis of the 16S rRNA gene sequence (1460 bp) of ZS110521T revealed that it displayed the highest similarity to Lentibacillus populi WD4L-1T (95.5 %), followed by Lentibacillus garicola SL-MJ1T (95.4 %) and Lentibacillus lacisalsi BH260T (95.2 %). ANI and dDDH values between ZS110521T and other strains of species of the genus Lentibacillus were less than 78 and 28 %, respectively. The predominant cellular fatty acids (> 10 %) of ZS110521T were anteiso-C17 : 0 (37.8 %), anteiso-C15 : 0 (28.1 %) and iso-C16 : 0 (15.5 %). The respiratory quinone was identified as menaquinone-7 (MK-7) and the major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The polyphasic taxonomic data and the results of chemotaxonomic analysis confirmed that ZS110521T represents a novel species, for which the name Lentibacillus daqui sp. nov. is proposed. The type strain of this proposed species is ZS110521T (=CGMCC 1.19456T =JCM 35213T).

Machine learning based age-authentication assisted by chemo-kinetics: Case study of strong-flavor Chinese Baijiu.

The aged Chinese liquor, Baijiu, is highly valued for its superior organoleptic qualities. However, since age-authentication method and aging-mechanism elucidation of Baijiu is still in the exploratory stage, high-quality aged Baijiu is often replaced by lower-quality, less-aged product with fraudulent mislabeling. Authentic high-quality strong-flavor Baijiu was analyzed by gas chromatography-mass spectrometry. Total esters decreased with aging, while acids, alcohols, aldehydes, ketones, terpenes, pyrazines increased. Although concentrations of partial compounds showed non-monotonic profiling during aging, a close positive linear correlation (R2 = 0.7012) of Baijiu Evenness index (0.55-0.59) with aging time was observed, indicating a more balanced composition in aged Baijiu. The reaction quotient (Qc) of each esterification, calculated by the corresponding reactant and product concentration, approached to the corresponding thermodynamic equilibrium constant Kc. This result demonstrated that the spontaneous transformation driven by thermodynamics explained part of the aging compositional profiling. Furthermore, an aging-related feature selection and an age-authentication method were established based on three models combined with five ranking algorithms. Forty-one key features, including thirty-six compound concentrations, four esterification Qc values and the Evenness index were selected out. The age-authentication based on neural network using forty-one input features accurately predicted the age group of Baijiu samples (F1 = 100 %). These findings have deepened understanding of the Baijiu aging mechanism and provided a novel, effective approach for age-authentication of Baijiu and other liquors.

Metabolomic analysis of the effects of a mixed culture of Saccharomyces cerevisiae and Lactiplantibacillus plantarum on the physicochemical and quality characteristics of apple cider vinegar.

Introduction: This study compared differences in physicochemical characteristics of the vinegar made by a mixed culture (MC) of Saccharomyces cerevisiae and Lactiplantibacillus plantarum and a pure culture (PC) of Saccharomyces cerevisiae. Methods: The fermentation process was monitored, and metabolomics analysis by Liquid Chromagraphy-Mass Spectrometry (LC-MS) was applied to the compositional differences between PC and MC vinegars, combined with quantification of organic acids, amino acids and B vitamins. Results: A total of 71 differential metabolites including amino acids, organic acids and carbohydrates, and six possible key metabolic pathways were identified. MC enhanced the malic acid utilization and pyruvate acid metabolism during fermentation, increasing substrate-level phosphorylation, and supplying more energy for cellular metabolism. Higher acidity at the beginning of acetic acid fermentation, resulting from lactic acid production by Lactiplantibacillus plantarum in MC, suppressed the cellular metabolism and growth of Acetobacter pasteurianus, but enhanced its alcohol metabolism and acetic acid production in MC. MC vinegar contained more vitamin B, total flavonoids, total organic acids, amino acids and had a higher antioxidant capacity. MC enhanced the volatile substances, particularly ethyl lactate, ethyl caprate and ethyl caproate, which contributed to a stronger fruity aroma. Discussion: These results indicated the mixed culture in alcoholic fermentation can effectively enhance the flavor and quality of apple cider vinegar.

Effect of Ambient Temperature on the Mechanical Properties of High Ductility Concrete.

This study analyzes the mechanical properties of high ductility concrete (HDC) under different ambient temperatures to provide a parameter basis for the design of HDC bridge deck link slabs. Five temperatures (-30, 0, 20, 40, and 60 °C) were designed to investigate the compressive, tensile, and flexural properties of HDC after temperature treatment and analyze the pore structure. The results show that, compared with the HDC performance at room temperature (20 °C), the compressive strength, ultimate tensile strength, and flexural strength decreased after treatment at low temperatures (-30 and 0 °C), while the strength increased after treatment at high temperatures (40 and 60 °C). After experiencing low- and high-temperature treatments, the ultimate tensile strain and ultimate deflection of the HDC increased. The tensile and flexural failures of the HDC exhibited multiple cracking, and the stress-strain/deflection curve showed a strain/deflection hardening stage. The tensile constitutive relationship can be simplified as a bilinear two-stage relationship. As the temperature increased, the porosity of harmless and less harmful pores in HDC gradually increased, while the porosity of harmful and more harmful pores gradually decreased, resulting in an increase in HDC strength. Based on the influence of temperature on HDC properties, design parameters for the HDC bridge deck link slab structure are proposed.

Notoginsenoside R1 inhibits hepatitis B virus replication by modulating SIRT1 activity.

The hepatitis B virus (HBV) infection remains highly prevalent globally. The present study aimed to explore the possible therapeutic effect of notoginsenoside R1, which has attracted considerable attention due to its diverse pharmacological effects, on HBV infection. The HBV-containing hepatocellular carcinoma cell lines, HepG2 and MHCC97H, were used in this study. We first treated the two cell lines with different concentrations of notoginsenoside R1 and subsequently measured the relative levels of HBV DNA, HBV surface antigen, HBV core antigen, and sirtuin 1 (SIRT1) using reverse transcription-quantitative polymerase chain reaction and western blotting. Finally, an HBV hemodynamic replication model was created to test the effect of notoginsenoside R1 on HBV replication. Notoginsenoside R1 inhibited the replication of HBV. This inhibitory effect was mediated through the downregulation of SIRT1 activity. Additionally, the inhibition of SIRT1 activity by silencing its expression or treatment with the SIRT1 inhibitor, selisistat, suppressed HBV replication. Furthermore, our animal experiments demonstrated that notoginsenoside R1 was effective at suppressing HBV replication in vivo. Thus, notoginsenoside R1 suppresses HBV replication by downregulating SIRT1 activity in vitro and in vivo. Keywords: notoginsenoside R1; hepatitis B virus; SIRT1.

Bacteria and filamentous fungi running a relay race in Daqu fermentation enable macromolecular degradation and flavor substance formation.

As the saccharifying and fermentative agent, medium-temperature Daqu (MT-Daqu) plays an irreplaceable role in the production of strong-flavor Baijiu. Numerous studies have focused on the microbial community structure and potential functional microorganisms, however, little is known about the succession of active microbial community and the formation mechanism of community function during MT-Daqu fermentation. In this study, we presented an integrated analysis of metagenomics, metatranscriptomics, and metabonomics covering the whole fermentation process of MT-Daqu to reveal the active microorganisms and their participations in metabolic networks. The results showed that dynamic of metabolites were time-specific, and the metabolites and co-expressed active unigenes were further classified into four clusters according to their accumulation patterns, with members within each cluster displaying a uniform and clear pattern of abundance across fermentation. Based on KEGG enrichment analysis in co-expression clusters and succession of active microbial community, we revealed that Limosilactobacillus, Staphylococcus, Pichia, Rhizopus, and Lichtheimia were metabolically active members at the early stage, and their metabolic activities were conducive to releasing abundant energy to drive multiple basal metabolisms such as carbohydrates and amino acids. Thereafter, during the high temperature period and at the end of fermentation, multiple heat-resistant filamentous fungi were transcriptionally active populations, and they acted as both the saccharifying agents and flavor compound producers, especially aromatic compounds, suggesting their crucial contribution to enzymatic activity and aroma of mature MT-Daqu. Our findings revealed the succession and metabolic functions of the active microbial community, providing a deeper understanding of their contribution to MT-Daqu ecosystem.

Volatile Compound Abundance Correlations Provide a New Insight into Odor Balances in Sauce-Aroma Baijiu.

Sauce-aroma Baijiu (SAB) is one of the most famous Baijius in China; SAB has more than 500 aroma compounds in it. However, the key aroma compound in SAB flavor remains unclear. Volatiles play an important role in SAB aroma and are highly correlated to SAB quality. In the present study, 63 volatile compounds were quantified among 66 SAB samples using gas chromatography with flame ionization detector (GC-FID). The authors analyzed odor contributions and volatile compound correlations in two quality groups of SAB samples. Moreover, an odor activity value (OAV) ratio-based random forest classifier was used to explain the volatile compound relationship differentiations between the two quality groups. Our results proved higher quality SABs had richer aromas and indicated a set of fruity-like ethyl valerate, green- and malt-like isobutyraldehyde and malt-like 3-methylbutyraldehyde and sweet-like furfural, had closer co-abundance correlations in higher quality SABs. These results indicated that the aroma and contributions of volatile compounds in SABs should be analyzed not only with compound odor activity values, but also the correlations between different aroma compounds.

Components of Salvia miltiorrhiza and Panax notoginseng Protect Pericytes Against OGD/R-Induced Injury via Regulating the PI3K/AKT/mTOR and JNK/ERK/P38 Signaling Pathways.

Salvia miltiorrhiza (SAL) and Panax notoginseng (PNS) are widely used in treating of ischemic stroke. However, it is unknown which components of SAL and PNS protect brain microvascular pericytes after an ischemic stroke. We evaluated the protective effects and mechanisms of SAL and PNS components in pericytes subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Pericytes were subjected to OGD/R. Cell Counting Kit-8 (CCK-8) was used to evaluate cell viability. ROS and SOD kits were used to detect oxidative stress. Flow cytometry was performed to analyze cell apoptosis. To evaluate cell migration, a scratch assay was performed. Expression of cleaved caspase-3, Bcl-2, Bax, VEGF, Ang-1, PDGFR-ß, PI3K/AKT/mTOR, and JNK/ERK/P38 signaling pathways were identified using western blot. The results revealed that salvianolic acid B (Sal B), salvianolic acid D (Sal D), notoginsenoside R1 (R1), ginsenoside Rb1 (Rb1), and ginsenoside Rg1 (Rg1) increased the cell viability of pericytes subjected to OGD/R, reduced the level of ROS, and increased the expression of SOD. The components reduced cell apoptosis, increased the protein level of Bcl-2/Bax, reduced the level of cleaved caspase-3/caspase-3, increased cell migration, and enhanced the levels of Ang-1, PDGFR-ß, and VEGF. The components could activate PI3K/AKT/mTOR pathway while inhibiting the JNK/ERK/P38 pathway. Studies found that Sal B, Sal D, R1, Rb1, and Rg1 inhibited oxidative stress and apoptosis while increasing the release of pro-angiogenic regulators of pericytes related to the PI3K/AKT/mTOR and JNK/ERK/P38 signaling pathways. This provides a potential foundation for developing monomeric drugs for treating ischemic stroke.

Neobavaisoflavone ameliorates LPS-induced RAW264.7 cell inflammations by suppressing the activation of NF-κB and MAPKs signaling pathways.

Objectives: Neobavaisoflavone (NBIF) is an isoflavone isolated from Psoralea corylifolia L. It can effectively regulate the redox state as a natural anti-oxidant and show some anti-inflammatory activity. However, its molecular mechanism is poorly studied. In this study, RAW264.7 cells were treated with lipopolysaccharide (LPS) to investigate the anti-inflammatory activity and potential NBIF mechanism. Materials and Methods: RAW264.7 cells were treated with LPS (62.5 ng/ml) and exposed to different concentrations of NBIF (0.01, 0.1, and 1 µM) for 24 hr. Inflammatory cytokines of RAW264.7 cells were measured by the Griess method, ELISA, and western blot. Phagocytosis of RAW264.7 macrophages was measured by FITC-dextran uptake assay. The phosphorylation protein expression levels of MAPKs (JNK, p38, and ERK), NF-κB p65, IκBα, and IκB kinase were analyzed by western blot. The results were analyzed using one-way ANOVA with Tukey's multiple comparison test. Results: NBIF significantly inhibited NO and ROS production by down-regulation of iNOS and COX-2 protein expression. Additionally, the amount of release and protein levels of inflammation cytokines IL-6, IL-1ß, and TNF-α were significantly decreased by NBIF. Moreover, FITC-dextran uptake assay by flow cytometry presented that NBIF significantly enhanced the phagocytic capacity of RAW264.7. Mechanistically, NBIF significantly down-regulated MAPK activation and inhibited the nuclear translocation of NF-κB p65. Conclusion: The present study demonstrates that NBIF inhibited inflammation and enhanced the phagocytic capacity of RAW264.7 cell-related MAPKs and NF-κB signaling pathways induced by LPS. These findings suggest that NBIF may have clinical utility as an anti-inflammatory agent.

Spatial heterogeneity of the microbiome and metabolome profiles of high-temperature Daqu in the same workshop.

High-temperature Daqu, usually used as a fermentation starter for sauce-flavor Baijiu production, plays an essential role in the yield and flavor quality of Baijiu. The environmental heterogeneity of different locations in the workshop during fermentation led to the final production of Daqu with three different types (i.e., white, yellow, and black Daqu). How to use these three types of Daqu in Baijiu production mainly depends on the workers' experience so far. Here, we aimed to reveal the potential functions of different types of Daqu by comparing enzyme activity, volatile metabolites, and microbiota characteristics. White_Qu exhibited the highest liquefaction and saccharification enzyme activities, while the highest neutral proteinase and cellulase enzyme activities were detected in black_Qu. The total volatile content of yellow_Qu and black_Qu was roughly double that of white_Qu, and multivariate analysis revealed distinct volatile dissimilarities across different types of Daqu. Significant differences in bacterial and fungal community structures, assembly patterns, and potential functional profiles were discovered among different types of Daqu. At the genus level, Oceanobacillus and Thermomyces dominated the white_Qu microbiota, and the abundant microbes in yellow_Qu and black_Qu were scattered in Kroppenstedtia and Thermoascus. Bacterial and fungal communities were dominated by deterministic and stochastic assembly processes, respectively, suggesting that bacteria may be more affected by abiotic environmental factors and species interaction than fungi. Co-occurrence network analysis showed positive correlations characterized Daqu microbial networks, and network topological features indicated stronger interactions between bacterial taxa compared with fungal community. The Spearman correlation analysis revealed that four bacterial genera (Kroppenstedtia, Virgibacillus, Scopulibacillus, and Staphylococcus) and two fungal genera (Thermoascus and Aspergillus) exhibited positive correlations with almost all of the abundant volatiles. This work reveals that spatially varying environments lead to the microbiome and metabolome heterogeneity of high-temperature Daqu in the same workshop.

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.

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

Clostridium 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.

Xueshuantong injection combined with Salvianolate lyophilized injection improves the synaptic plasticity against focal cerebral ischemia/reperfusion injury in rats through PI3K/AKT/mTOR and RhoA/ROCK pathways.

The combined use of two or more different drugs can better promote nerve recovery and its prognosis for treatment of stroke. Salvianolate lyophilized injection (SLI) made from the aqueous extraction of salvia miltiorrhiza and Xueshuantong injection (lyophilized) (XST) made from the Panax Notoginseng extraction are two herbal standardized preparations that have been widely used in China for the treatment of ischemic stroke. In this study, we investigated the neuroprotective effects of XST combined with SLI in the recovery stage of middle cerebral artery occlusion / reperfusion (MCAO/R) injury rat. Wistar rats were subjects to MCAO/R, then were treated with SLI or XST alone, or with their combination (1X1S) via tail injection daily for 14 days. The pathological status of the brain was detected by neurological deficit scores, TTC, regional cerebral blood flow and Nissl staining. Golgi-Cox staining was used to assess dendritic, axonal and synaptic remodeling. The expression of MAP-2, ß-Tubulin, PSD95, SYN, BDNF and VEGF were analyzed by western blotting and immunofluorescence. The results showed that administration of 1X1S not only significantly decreased neurological scores and infarct volumes, but also increased regional cerebral blood flow, strengthened dendritic and synaptic remodeling compared with XST, SLI used alone. And the mechanism of combined of 1X1S to exert neuroprotection may be associated with PI3K/ AKT/ mTOR and RhoA/ROCK2 pathways. Overall, these findings suggest that combination of XST and SLI promotes dendritic spine density and synaptic plasticity via upregulation of the PI3K/ AKT/ mTOR pathways and inhabitation the RhoA/ROCK2 signaling pathway in rat with MCAO/R, showing its multiple-action-multiple-target efficacy and suggest a potential new strategy for ischemia.

Combined effects of fermentation starters and environmental factors on the microbial community assembly and flavor formation of Zhenjiang aromatic vinegar.

Microbial ecosystems of fermented foods are largely interfered by human activities in myriad ways. The aim of this study was to illuminate the impacts of various starters and environmental variables on the fermentation process of Zhenjiang aromatic vinegar (ZAV), one of the four representative cereal vinegars in China. The effects of environmental variables (e.g., ethanol, total acidity, temperature) and starters (e.g., jiuqu, maiqu, seed pei) on the profiles of microbiome and metabolome (e.g., organic acids, amino acids and volatiles) during fermentation process of ZAV were analyzed. Amongst the four fermentation stages, acetic acid fermentation was the main stage for the accumulation of flavor substances, and subsequently, the contents of acids (mainly acetic, lactic and citric acids) and volatile metabolites (e.g., 2,3-butanedione, acetoin, etc.) continued to enrich in sealed fermentation stage. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) showed that the fungal and bacterial community structures of four fermentation stages were significantly different. As for bacterial community, the dominant OTUs with average relative abundance over 10% in at least one fermentation stage were assigned to the genera Acetilactobacillus, Acetobacter, Acinetobacter, Aeromonas, Lactobacillus, and Pseudomonas. The dominant fungal populations in each fermentation stage were obviously divergent, including Wickerhamomyces, Saccharomyces, Alternaria, Fusarium, etc. SourceTracker analysis demonstrated that jiuqu and seed pei provided microorganisms to initiate starch saccharification and acetic acid fermentation stages, respectively, and maiqu was mainly the donor of enzymes in alcohol fermentation. Spearman correlation coefficients revealed positive relationships between fungal community and various flavor metabolites, indicating the essential role of fungi in the flavor formation of ZAV. This study systematically reveals the effects of fermentation starters and environmental variables on vinegar production and deepens the understanding of the traditional production craft.