Bioturbation analysis of microbial communities and flavor metabolism in a high-yielding cellulase Bacillus subtilis biofortified Daqu.

In this study, a fortified Daqu (FF Daqu) was prepared using high cellulase-producing Bacillus subtilis, and the effects of in situ fortification on the physicochemical properties, flavor, active microbial community and metabolism of Daqu were analyzed. The saccharification power, liquefaction power, and cellulase activity of the FF Daqu were significantly increased compared with that of the traditional Daqu (CT Daqu). The overall differences in flavor components and their contents were not significant, but the higher alcohols were lower in FF Daqu. The relative abundance of dominant active species in FF Daqu was 85.08% of the total active microbiota higher than 63.42% in CT Daqu, and the biomarkers were Paecilomyces variotii and Aspergillus cristatus, respectively. The enzymes related to starch and sucrose metabolic pathways were up-regulated and expressed in FF Daqu. In the laboratory level simulation of baijiu brewing, the yield of baijiu was increased by 3.36% using FF Daqu.

Combined microbiome and metabolomics analysis of Taorong-type baijiu high-temperature Daqu and medium-temperature Daqu.

Background: Daqu is an essential starter for baijiu brewing in China. However, the microbial enrichment and metabolic characteristics of Daqu formed at different fermentation temperatures are still unclear. Methods: High-throughput sequencing technology and the non-targeted metabolomics were used to compare the microbial communities and metabolites of Taorong-type high-temperature Daqu and middle-temperature Daqu. In this study, the relationship between microorganisms and metabolites was established. Results: The study found that the composition and metabolites of the microbial community differed due to the difference in Daqu-making temperature. The bacterial diversity of Taorong-type high-temperature Daqu was higher than that of middle-temperature Daqu, while the fungal community diversity of Taorong-type middle-temperature Daqu was higher than that of high temperature Daqu. A total of 1,034 differential metabolites were screened from the two types of Daqu, and 76 metabolites with significant differences were detected (P < 0.001 and variable importance in projection (VIP) > 1.15). Tetraacetylethylenediamine is the metabolite with the largest differential fold among the 76 differential metabolites, which can be used as a potential marker metabolite of high-temperature Daqu. Conclusion: This study helps elucidate the microbial assembly mechanisms and functional expression under different processing conditions through a further understanding of the composition and metabolic profile differences of different types of Daqu microflora in Taorong-type baijiu.

Screening and identification of high yield tetramethylpyrazine strains in Nongxiangxing liquor Daqu and study on the mechanism of tetramethylpyrazine production.

BACKGROUND: There are few reports on the breeding of high-yielding tetramethylpyrazine (TTMP) strains in strong-flavor Daqu. In addition, studies on the mechanism of TTMP production in strains are mostly based on common physiological and biochemical indicators, and there is no report on RNA level. Therefore, in this study, a strain with high production of TTMP was screened out from strong-flavor liquor, and transcriptome sequencing analysis was performed to analyze its key metabolic pathways and key genes, and to infer the mechanism of TTMP production in the strain. RESULTS: In this study, a strain with a high yield of tetramethylpyrazine (TTMP) was screened out, and the yield was 29.83 µg mL-1 . The identified strain was Bacillus velezensis, which could increase the content of TTMP in liquor by about 88%. After transcriptome sequencing, a total of 1851 differentially expressed genes were screened, including 1055 up-regulated genes and 796 down-regulated genes. Three pathways related to the production of TTMP were identified by gene ontology (GO) annotation and COG annotation, including carbohydrate metabolism, cell movement and amino acid metabolism. The key genes of TTMP were analyzed, and the factors that might regulate the production of TTMP, such as the transfer of uracil phosphate ribose and glycosyltransferase, were obtained. CONCLUSIONS: A strain of B. velezensis with high TTMP production was screened and identified in strong-flavor Daqu for the first time. The yield of TTMP was 29.83 µg mL-1 , which increased the TTMP content in liquor by 88%. The key metabolic pathways of TTMP production in the strain were obtained: carbohydrate metabolism, cell movement and amino acid metabolism, and the key regulatory genes of each pathway were found, which complemented the gap in gene level in the production regulation of the strain, and provided a theoretical basis for the subsequent study of TTMP in liquor. © 2023 Society of Chemical Industry.

Optimization of fermentation technology for composite fruit and vegetable wine by response surface methodology and analysis of its aroma components.

Fruit wine has certain health care functions, but fruit wine made from a single fruit or vegetable does not have a good enough color, flavor or nutrient composition. Therefore, this study used fresh carrot (Daucus carota subsp. sativus) and pomegranate (Punica granatum) as raw materials to explore the brewing process of carrot and pomegranate compound wine. The fermentation technology of the composite carrot and pomegranate wine was optimized by a single-factor experiment and Box-Behnken design (BBD), which provided a theoretical foundation for the fermentation of this wine. As per the results, the alcohol content of this composite carrot and pomegranate wine was 12.35% vol. under the optimum fermentation conditions of 28 °C initial temperature, 24% initial sugar content, and with the addition of 64 mg L-1 sulfur dioxide (SO2). In the fermented fruit and vegetable wine, a total of 30 aroma components were detected; 21 composites (such as bornyl acetate, caryophyllene and 3-(2-nitrophenylmethyl)-2-thiazolidinone) were newly generated. The relative content of alcohol flavor composites (such as propionic acid 2-methyl-3-hydroxy-2,2,4-trimethylpentan-1-ol, 2-methyl-2-ethyl-3-hydroxycyclohexyl propanoate and terpinene-4-ol) showed an upward trend, and the relative content of alkene components increased significantly after fermentation. The findings of this study provide an experimental foundation for optimizing fermentation technology and for improving the product quality of composite carrot and pomegranate wine.

Taorong-type Baijiu starter: Analysis of fungal community and metabolic characteristics of middle-temperature Daqu and high-temperature Daqu.

To study the difference between the fungal community compositional and fragrance components in medium- and high-Temperature Taorong-type Baijiu Daqu. The microbial communities and fragrance components of Taorong-type Baijiu Daqu were analyzed using high-throughput sequencing (HTS) and headspace-solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). With an abundance at the phylum and genus levels ≥0.01% as the threshold, 3 phyla, Mucoromycota, Ascomycota, and Basidiomycota, were found in both medium- and high-temperature Daqu, but their abundances differed. At the genus level, 15 and 13 genera were recognized. Rhizopus (72.40%) and Thermomyces (53.32%) accounted for the most significant proportions in medium-temperature and high-temperature Daqu, respectively. Medium-temperature Daqu and high-temperature Daqu were found to have 40 and 29 fragrance components, respectively and contained the highest proportions of pyrazines (53.12%) and acids (32.68%). Correlation analyses between microbes and fragrance components showed that Aspergillus, Hyphopichia, Trichosporon, Alternaria were all highly and positively correlated with pyrazines, but the dominant fungal communities were highly correlated with only a few individual acid compounds but not with acid compounds overall. The unique Daqu -making process and environment lead to these differences.

Compositional Alterations of the Nasal Microbiome and Staphylococcus aureus-Characterized Dysbiosis in the Nasal Mucosa of Patients With Allergic Rhinitis.

OBJECTIVES: Host-microbial commensalism can shape the innate immune response in the nasal mucosa, and the microbial characteristics of nasal mucus directly impact the mechanisms of the initial allergic responses in the nasal epithelium. We sought to determine alterations of the microbial composition in the nasal mucus of patients with allergic rhinitis (AR) and to elucidate the interplay between dysbiosis of the nasal microbiome and allergic inflammation. METHODS: In total, 364,923 high-quality bacterial 16S ribosomal RNA-encoding gene sequence reads from 104 middle turbinate mucosa samples from healthy participants and patients with AR were obtained and analyzed using the Quantitative Insights into Microbial Ecology pipeline. RESULTS: We analyzed the microbiota in samples of nasal mucus from patients with AR (n=42) and clinically healthy participants (n=30). The Proteobacteria (Ralstonia genus) and Actinobacteria (Propionibacterium genus) phyla were predominant in the nasal mucus of healthy subjects, whereas the Firmicutes (Staphylococcus genus) phylum was significantly abundant in the nasal mucus of patients with AR. In particular, the Ralstonia genus was significantly dominant in the clinically healthy subjects. Additional pyrosequencing data from 32 subjects (healthy participants: n=15, AR patients: n=17) revealed a greater abundance of Staphylococcus epidermidis, Corynebacterium accolens, and Nocardia coeliaca, accounting for 41.55% of mapped sequences in the nasal mucus of healthy participants. Dysbiosis of the nasal microbiome was more pronounced in patients with AR, and Staphylococcus aureus exhibited the greatest abundance (37.69%) in their nasal mucus, in association with a positive response to house dust mites and patients' age and height. CONCLUSION: This study revealed alterations in the nasal microbiome in the nasal mucus of patients with AR at the levels of microbial genera and species. S. aureus-dominant dysbiosis was distinctive in the nasal mucus of patients with AR, suggesting a role of host-microbial commensalism in allergic inflammation.

Comprehensive evaluation of Luzhou-flavor liquor quality based on fuzzy mathematics and principal component analysis.

Currently, the primary method of identifying high- and low-quality liquors is sensory tasting, which is prone to uncertainty caused by the biases of tasters. To address this problem, this study used color, aroma, taste, and style as four factors affecting the sensory quality of Luzhou-flavor liquor; determined the weights of each factor; and quantitatively evaluated the sensory quality of five different Luzhou flavor liquor using fuzzy mathematical methods. The volatile aromatic substances in the liquor samples were detected by GC-MS, and analyzed using principal component analysis. The results obtained from fuzzy mathematics and principal component analysis indicated that the comprehensive evaluation system was scientifically sound and reasonably constructed.

Analysis of microbial community structure and volatile compounds in pit mud used for manufacturing Taorong-type Baijiu based on high-throughput sequencing.

In this study, the pit mud used in manufacturing Taorong-type Baijiu was collected from the upper, middle, lower and bottom layers of pits at Henan Yangshao Liquor Co., LTD. High-throughput sequencing (HTS) technology was used to analyze the microbial community structure of the pit mud. In addition, the volatile compounds in the pit mud were subjected to preliminary qualitative analysis through headspace-solid phase microextraction and gas chromatography-mass spectrometry (GC-MS). The HTS results demonstrated that there were 5, 3, 5 and 5 dominant bacterial phyla (including 11, 11, 9 and 8 dominant bacterial genera) and 3, 3, 3 and 3 dominant fungal phyla (including 4, 7, 7 and 5 dominant fungal genera) in the pit mud from the F-S (upper), G-Z (middle), H-X (lower) and I-D (bottom) layers, respectively. In the qualitative analysis of the volatile compounds, a total of 77types of volatile compounds were detected in the pit mud, including 46, 45, 39 and 49 types in the pit mud from layers F-S, G-Z, H-X and I-D, respectively. Esters and acids were the two main components of the pit mud. The correlation between the microorganisms present and the main volatile compounds in the pit mud was analyzed. Lentimicrobium, Syner-01 and Blvii28_wastewater-sludge groups were found for the first time in pit mud used for manufacturing Taorong-type Baijiu. The findings of this study could provide a theoretical foundation for improving the quality of pit mud and the flavor of Taorong-type Baijiu.

Isolation, identification, and whole-genome sequencing of high-yield protease bacteria from Daqu of ZhangGong Laojiu.

A total of 296 strains of protease-producing bacteria were isolated and purified from medium-temperature Daqu produced by ZhangGong LaoJiu Wine Co. Ltd. After calculating the ratio of transparent ring diameter to colony diameter and measuring the protease activities, a strain of high-yield protease bacteria, called DW-7, was screened out with a protease activity of 99.54 U/mL. Through morphological observation, 16S rDNA sequence analysis, and physiological and biochemical tests, the isolated bacteria DW-7 was determined to be Bacillus velezensis. In addition, whole-genome sequencing (WGS), using PacBio and the Illumina platform, was performed. Gene annotation was then conducted using the Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Non-Redundant Protein Sequence Database (NR), and Gene Ontology (GO) databases. The results showed that the genome of DW-7 was 3,942,829 bp long with a GC content of 46.45%. A total of 3,662 protein-encoding genes were predicted, with a total length of 3,402,822 bp. Additionally, 2,283; 2,796; and 2,127 genes were annotated in the COG, KEGG, and GO databases, respectively. A total of 196 high-yield protease genes were mainly enriched in the metabolism of alanine, aspartic acid, glutamate, glycine, serine, and threonine, as well as ABC transporter and transporter pathways.

Profiling the composition and metabolic activities of microbial community in fermented grain for the Chinese strong-flavor Baijiu production by using the metatranscriptome, high-throughput 16S rRNA and ITS gene sequencings.

The composition and function of microbial community analyzed by sequencing 16S rRNA/ITS gene amplicons (DNA level) were compared with those derived by using metatranscriptome sequencing (RNA level) from the same fermented grain (FG) sample, which obtained from the key fermentation time point during the Chinese strong-flavor Baijiu (CSFB) production process. The results showed that the fungi with the highest relative abundance was Saccharomyces (RNA: 83.15%, DNA: 89.74%) at the two levels. The most abundant bacterium was Kroppenstedtia (37.09%) detected only at the DNA level, while it was Streptococcus (93.75%) at the RNA level, indicating that the structures of prokaryotic communities at the two levels were quite different. For the microbial functions, a large proportion of genes of FG microorganisms related to "Metabolism" function were observed both by using PICRUSt2 analysis (DNA level) and metatranscriptome analysis (RNA level), and especially enriched in "Carbohydrate metabolism". While the proportions of genes involved in some functions were different, such as "Replication and repair", "Membrane transport" and "Environmental adaptation", with high proportions of genes involved in at the DNA level when compared those at the RNA level. Furthermore, Saccharomyces cerevisiae was the most active microbe in the top15 pathways, followed by Torulaspora dellbrueckii. During the conversion of starch to ethanol, S. cerevisiae showed high metabolic capacity, and cooperated with other microorganisms to convert pyruvate to acetaldehyde directly or through acetyl-CoA and acetate, and then acetaldehyde to ethanol. As far as we know, this is a first study to profile the microbial community and metabolic features in FG of CSFB by using a combination of DNA- and RNA- based technologies. Our findings could provide useful insights for further understanding the active microbial function, metabolic pathways and fermentation mechanism in the FG ecosystem during CSFB fermentation.

Cu-Catalyzed Asymmetric Hydroboration of Naphthylallylic Compounds for Enantioselective Synthesis of Chiral Boronates.

A Cu-catalyzed regio- and enantioselective hydroboration of various naphthylallylic compounds affording chiral boronates with high yields and excellent enantioselectivities (up to 96% ee) was presented. The utility of the boronated products is further illustrated by other stereospecific C-B bond transformations to produce amino alcohols and other useful compounds.

Enantioselective Synthesis of Boryl Tetrahydroquinolines via Cu-Catalyzed Hydroboration.

A Cu-catalyzed regio- and enantioselective hydroboration of 1,2-dihydroquinolines with high yields and excellent enantioselectivities (up to 98% ee) was presented. This method could be applied in the asymmetric synthesis of the important intermediates used in the enantioselective synthesis of the potential agent Sumanirole for the treatment of Parkinson's disease and of the potentially interesting positive inotropic agent (S)-903.

Kinetic resolution of racemic 2-substituted 1,2-dihydroquinolines via asymmetric Cu-catalyzed borylation.

A highly efficient kinetic resolution of racemic 2-substituted 1,2-dihydroquinolines via asymmetric Cu-catalyzed borylation has been realized for the first time. Under mild conditions, a variety of chiral 3-boryl-1,2,3,4-tetrahydroquinolines containing two vicinal stereogenic centers as well as the recovered 2-substituted 1,2-dihydroquinolines were afforded after 30 minutes in high yields with up to 99% ee (dr > 99 : 1) and over 98% ee values, respectively, corresponding to kinetic selectivity factors of up to 569. Moreover, this protocol was successfully applied to the asymmetric synthesis of a selective estrogen receptor modulator.