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Abstract 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.

ABSTRACT The fermentation process of strong-flavor Baijiu occurs in an underground cellar surrounded by mud, where grains are decomposed and generate new flavor substances. Pit mud microbiome plays a pivotal role in the formation of Baijiu flavor, which is positively correlated with cellar age. Previous research on the pit mud microbiome was often conducted for a specific distillery, making it hard to draw a general conclusion to decipher the pit mud microecosystem in such a unique brewing environment. Hence, in this study, we attempt to unravel community formation mechanisms through research in pit mud microbial biogeography based on 12 independent bacterial studies comprising qualified data sets of 302 samples clearly marked with cellar age from five major producing provinces. We found cellar age explained more variations in the bacterial community than geographical region. The bacterial community displayed evident dominant species alternations on a large time scale. Lactobacillus was found to be the dominant species in young pit mud (

IntroductionThis 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.MethodsThe 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.ResultsA 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.DiscussionThese results indicated the mixed culture in alcoholic fermentation can effectively enhance the flavor and quality of apple cider vinegar.

ABSTRACT 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.

Cereal vinegar sediment (CVS) is a natural precipitate formed during the aging process of traditional grain vinegar. It has been used as Chinese traditional medicine, while its composition and function are reported minimally. In this study, we measured CVS in terms of saccharide, protein, fat and water content, and polyphenol and flavonoid content. Furthermore, we determined the amino acids, organic acids, and other soluble metabolites in CVS using reverse-phase high-performance liquid chromatography (RP-HPLC), HPLC, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) platforms. The hepatoprotective effect of CVS was evaluated in acute CCl4-induced liver injury mice. Administration of CVS for 7 days prior to the CCl4 treatment can significantly decrease liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and reactive oxygen species (ROS) levels, compared with those in the hepatic injury model group. The gut microbiota was changed by CCl4 administration and was partly shifted by the pretreatment of CVS, particularly the Muribaculaceae family, which was increased in CVS-treated groups compared with that in the CCl4 administration group. Moreover, the abundances of Alistipes genus and Muribaculaceae family were correlated with the liver ALT, AST, and malondialdehyde (MDA) levels. Our results illustrated the composition of CVS and its hepatoprotective effect in mice, suggested that CVS could be developed as functional food to prevent acute liver injury.

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.

Shuxuetong injection composed of leech (Hirudo nipponica Whitman) and earthworm (Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells (bEnd.3) were incubated in glucose-free Dulbecco’s modified Eagle’s medium containing 95% N2/5% CO2 for 6 hours, followed by high-glucose medium containing 95% O2 and 5% CO2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations (diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.

Butyrate, one of the key aroma compounds in Luzhou-flavor baijiu, is synthesized through two alternative pathways: butyrate kinase (buk) and butyryl-CoA: acetate CoA-transferase (but). A lack of knowledge of butyrate-producing microorganisms hinders our ability to understand the flavor formation mechanism of baijiu. Here, temporal dynamics of microbial metabolic profiling in fermented grains (FG) was explored via PICRUSt based on 16S rRNA gene sequences. We found Bacilli and Bacteroidia were the major potential butyrate producers in buk pathway at the beginning of fermentation, while later Clostridia dominated the two pathways. Clone library analysis also revealed that Clostridia (~73% OTUs) was predominant in buk pathway throughout fermentation, followed by Bacilli and Bacteroidia, and but pathway was merely possessed by Clostridia. Afterward, Clostridia-specific 16S rRNA gene sequencing demonstrated Clostridium might be the major butyrate-producing genus in two pathways, which was subsequently evaluated using culture approach. Seventeen Clostridium species were isolated from FG based on 16S rRNA gene sequence-guided medium prediction method. Profiles of short-chain fatty acids and but and buk genes in these species demonstrated phylogenetic and functional diversities of butyrate-producing Clostridium in FG. These findings add to illustrate the diversity of potential butyrate producers during brewing and provide a workflow for targeting functional microbes in complex microbial community.

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/mediumchain 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 b-oxidation. This work demonstrates the potential roles of mutually beneficial interspecies interactions in the accumulation of key flavor compounds in pit mud. [ABSTRACT FROM AUTHOR]

Female fig (Ficus carica L.) fruit are characterized by a major increase in volume and sugar content during the final week of development. A detailed developmental analysis of water and dry matter accumulation during these final days indicated a temporal separation between the increase in volume due to increasing water content and a subsequent sharp increase in sugar content during a few days. The results present fig as an extreme example of sugar import and accumulation, with calculated import rates that are one order of magnitude higher than those of other sugar-accumulating sweet fruit species. To shed light on the metabolic changes occurring during this period, we followed the expression pattern of 80 genes encoding sugar metabolism enzymes and sugar transporter proteins identified in fig fruit. A parallel comparison with male fig fruits, which do not accumulate sugar during ripening, highlighted the genes specifically related to sugar accumulation. Tissue-specific analysis indicated that the expression of genes involved in sugar metabolism and transport undergoes a global transition. [ABSTRACT FROM AUTHOR]

To obtain the design parameters of bridge deck link slabs made of ecological high-ductility cementitious composite (Eco-HDCC) material reinforced with a basalt fiber-reinforced plastic (BFRP) by applying fracture mechanics concepts, the reinforcing effects of a BFRP bar on the fracture properties of BFRP bar-reinforced Eco-HDCC specimens were studied. First, cracking load, crack mouth opening displacement (CMOD), and deflection of specimens were monitored, and BFRP bar diameter and cover thickness were considered. The results indicate that both the diameter and cover thickness of BFRP bar have an obvious influence on the crack kinking angle. In addition, as the diameter of BFRP bar increases, the peak load, peak CMOD, peak deflection, and fracture energy of Eco-HDCC specimens increase, and these peak values of Eco-HDCC specimens after reloading also show the same trend. In addition, the peak load, peak CMOD, peak deflection, and fracture energy are larger if the Eco-HDCC specimen has a smaller cover thickness. However, the Eco-HDCC specimen with a BFRP bar cover thickness of 25 mm has a higher peak load after reloading than the specimen with a cover thickness of 15 mm when the bar diameters are 10 and 12 mm, respectively. In conclusion, BFRP bar diameters from 10 to 14 mm and a cover thickness of 25 mm are recommended when designing bridge deck link slabs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Mature vinegar culture has usually been used as a type of autochthonous starter to rapidly initiate the next batch of acetic acid fermentation (AAF) and maintain the batch-to-batch uniformity of AAF in the production of traditional cereal vinegar. However, the vitality and dominance of functional microbes in autochthonous starters remain unclear, which hinders further improvement of fermentation yield and production. Here, based on metagenomic (MG), metatranscriptomic (MT), and 16S rRNA gene sequencings, 11 bacterial operational taxonomic units (OTUs) with significant metabolic activity (MT/MG ratio .1) and dominance (relative abundance .1%) were targeted in the autochthonous vinegar starter, all of which were assigned to 4 species (Acetobacter pasteurianus, Lactobacillus acetotolerans, L. helveticus, Acetilactobacillus jinshanensis). Then, we evaluated the successions and interactions of these 11 bacterial OTUs at different AAF stages. Last, a defined starter was constructed with 4 core species isolated from the autochthonous starter (A. pasteurianus, L. acetotolerans, L. helveticus, Ac. jinshanensis). The defined starter culture could rapidly initiate the AAF in a sterile or unsterilized environment, and similar dynamics of metabolites (ethanol, titratable acidity, acetic acid, lactic acid, and volatile compounds) and environmental indexes (temperature, pH) of fermentation were observed as compared with that of autochthonous starter (P . 0.05). This work provides a method to construct a defined microbiota from a complex system while preserving its metabolic function. IMPORTANCE Complex microorganisms are beneficial to the flavor formation in natural food fermentation, but they also pose challenges to the mass production of standardized products. It is attractive to construct a defined starter to rapidly initiate fermentation process and significantly improve fermentation yield. This study provides a comprehensive understanding of vital and dominant species in the autochthonous vinegar starter via multi-omics, and designs a defined microbial community for the efficient fermentation of cereal vinegar. [ABSTRACT FROM AUTHOR]

Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people's health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera (Staphylococcus, Bacillus, Weissella, Aspergillus, and Zygosaccharomyces) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function. [ABSTRACT FROM AUTHOR]