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).

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).

Constructing a Defined Starter for Multispecies Vinegar Fermentation via Evaluation of the Vitality and Dominance of Functional Microbes in an Autochthonous Starter.

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.

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.

Extreme sugar accumulation in late fig ripening is accompanied by global changes in sugar metabolism and transporter gene expression.

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.

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

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

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

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

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

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

Daqu microbiota exhibits species-specific and periodic succession features in Chinese baijiu fermentation process.

Daqu, a brick-shaped product spontaneously fermented under an open environment, has been regarded as the starter of fermentation, raw enzyme preparation and raw materials for baijiu production. However, its contribution in baijiu fermentation has not been fully elaborated yet. Here, the effects of daqu microbiota on baijiu fermentation were investigated under both field-scale and lab-scale conditions. In field-scale baijiu fermentation, the dominant daqu microbes (average relative abundance>10.0%), including unclassified_Leuconostocaceae, Thermoascus, and Thermomyces, tended to dominate the early stage (0-7 d). However, the rare daqu microbes (average relative abundance <0.1%, e.g., Kazachstania) tended to dominate the middle and late stages (11-40 d). In addition, some genera showed differences in species diversity between daqu and fermented grains. The average relative abundance of Lactobacillus was over 75% during baijiu fermentation, and most of them were affiliated with Lactobacillus acetotolerans, while Lactobacillus crustorum dominated the Lactobacillus OTUs in daqu. The similar patterns were also observed during lab-scale baijiu fermentation. The results of function prediction showed the enriched metabolic pathways were associated with glycolysis and long-chain fatty acid esters in baijiu fermentation. These results improved the understanding of daqu microbiota function during baijiu fermentation and provided a basic theory to support the regulation of baijiu production.

A Bottom-Up Approach To Develop a Synthetic Microbial Community Model: Application for Efficient Reduced-Salt Broad Bean Paste Fermentation.

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.IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment.

Lactobacillus jinshani sp. nov., isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar.

A novel Gram-stain-positive, non-motile, non-spore-forming, rod-shaped, facultatively anaerobic, designated strain HSLZ-75T, was isolated from the solid-state vinegar culture of Zhenjiang aromatic vinegar. Strain HSLZ-75T grew at 20-40 °C (optimum 35 °C), pH 3.0-5.0 (optimum pH 4.0) and 0-5% (w/v) NaCl (optimum 0%). Heterolactic fermentation characterised the metabolism of strain HSLZ-75T. D- and L-lactic acid were produced from glucose in a ratio of 91:9. The major cellular fatty acids ( > 10%) consisted of C16:0, C18:1ω9c, summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, one unidentified aminophospholipid, one unidentified phospholipid and six unknown lipids. The cell wall was found to contain meso-diaminopimelic acid-type peptidoglycan. The 16S rRNA gene sequence of strain HSLZ-75T showed the highest similarity of 88.0% with Lactobacillus fructivorans DSM 20203T. Phylogenetic analysis indicated that strain HSLZ-75T belonged to family Lactobacillaceae and formed a distinct lineage with the type strain of Lactobacillus caviae. The complete genome of strain HSLZ-75T contained a circular chromosome of 1,616,430 bp with 1570 genes and 39.7 mol% G + C content. The average nucleotide identity values between strain HSLZ-75T and the reference type strains Lactobacillus fructivorans DSM 20203T and Lactobacillus rossiae DSM 15814T were 66.4% and 65.7%, respectively. On the basis of phenotypic, chemotaxonomic, phylogenetic and genotypic characteristics, strain HSLZ-75T should be classified as a novel species of the genus Lactobacillus in the family Lactobacillaceae of the order Lactobacillales, for which the name Lactobacillus jinshani sp. nov. is proposed. The type strain is HSLZ-75T ( = CICC 6269T = JCM 33270T).

Deciphering the d-/l-lactate-producing microbiota and manipulating their accumulation during solid-state fermentation of cereal vinegar.

Symphony orchestra of multi-microorganisms characterizes the solid-state acetic acid fermentation process of Chinese cereal vinegars. Lactate is the predominant non-volatile acid and plays indispensable roles in flavor formation. This study investigated the microbial consortia driving the metabolism of D-/l-lactate during fermentation. Sequencing analysis based on D-/l-lactate dehydrogenase genes demonstrated that Lactobacillus (relative abundance: > 95%) dominated the production of both d-lactate and l-lactate, showing species-specific features between the two types. Lactobacillus helveticus (>65%) and L. reuteri (~80%) respectively dominated l- and d-lactate-producing communities. D-/l-lactate production and utilization capabilities of eight predominant Lactobacillus strains were determined by culture-dependent approach. Subsequently, D-/l-lactate producer L. plantarum M10-1 (d:l ≈ 1:1), l-lactate producer L. casei 21M3-1 (D:L ≈ 0.2:9.8) and D-/l-lactate utilizer Acetobacter pasteurianus G3-2 were selected to modulate the metabolic flux of D-/l-lactate of microbial consortia. The production ratio of D-/l-lactate was correspondingly shifted coupling with microbial consortia changes. Bioaugmentation with L.casei 21M3-1 merely enhanced l-lactate production, displaying ~4-fold elevation at the end of fermentation. Addition of L.plantarum M10-1 twice increased both D- and l-lactate production, while A. pasteurianus G3-2 decreased the content of D-/l-isomer. Our results provided an alternative strategy to specifically manipulate the metabolic flux within microbial consortia of certain ecological niches.

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

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

Xueshuantong injection (lyophilized) combined with salvianolate lyophilized injection protects against focal cerebral ischemia/reperfusion injury in rats through attenuation of oxidative stress.

Salvianolate lyophilized injection (SLI) and Xueshuantong injection (lyophilized) (XST) are two herbal standardized preparations that have been widely used in China for the treatment of acute cerebral infarction. In this study, we investigated the neuroprotective effects of SLI combined with XST in a rat model of middle cerebral artery occlusion-reperfusion (MCAO/R). Wistar rats were subjected to 1.5 h of MCAO followed by reperfusion for 3 h, then were treated with SLI or XST alone, or with their combinations via tail vein injection daily for 3 d. Edaravone (EDI, 6 mg·kg-1·d-1) was used as a positive control drug, We showed that administration of a combination of 1X1S (XST 100 mg·kg-1·d-1 plus SLI 21 mg·kg-1·d-1) more effectively protected the ischemic brains than SLI or XST used alone. Administration of 1X1S not only significantly decreased neurological deficit scores and infarct volumes and increased regional cerebral blood flow, but also inhibited the activation of both microglia and astrocytes in the hippocampus. Furthermore, administration of 1X1S significantly decreased the levels of MDA and ROS with concomitant increases in the levels of antioxidant activity (SOD, CAT and GSH) in the brain tissues as compared with SLI and XST used alone. Moreover, administration of 1X1S remarkably upregulated the expression of Nrf-2, HO-1 and NQO-1, and downregulated the expression of Keap1 and facilitated the nuclear translocation of Nrf-2 in the brain tissues as compared with XST used alone. Our study demonstrates that a combination of 1X1S effectively protects MCAO/R injury via suppressing oxidative stress and the Nrf-2/Keap1 pathway.

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.

Decoding the Qu-aroma of medium-temperature Daqu starter by volatilomics, aroma recombination, omission studies and sensory analysis.

Qu-aroma is of great significance for evaluation the quality of Daqu starter. This study aimed to decode the Qu-aroma of medium-temperature Daqu (MT-Daqu) via "top-down" and "bottom-up" approaches. Firstly, 52 aroma descriptors were defined to describe the MT-Daqu aroma by quantitative descriptive analysis. Secondly, 193 volatile organic compounds (VOCs) were identified from 42 MT-Daqu samples by HS-SPME-GC-MS, and 43 dominant VOCs were screened out by frequence of occurrence or abundance. By Thin Film (TF)-SPME-GC-O-MS, 27 odors and 90 VOCs were detected in MT-Daqu mixture, and 14 odor-active VOCs were screened out by odor intensity. Thirdly, a five-level MT-Daqu aroma wheel was constructed by matching 52 aroma descriptors and 37 aroma-active VOCs. Finally, Qu-aroma of MT-Daqu was reconstructed with 37 aroma-active VOCs and evaluated by omission experiments. Hereinto, 26 key aroma-active VOCs were determined by OAV value ≥1, including isovaleric acid, 1-hexanol, isovaleraldehyde, 2-octanone, trimethylpyrazine, γ-nonalactone, 4-vinylguaiacol, etc.

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.

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.

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.