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).

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).

Isolation and Characterization of Yeast with Benzenemethanethiol Synthesis Ability Isolated from Baijiu Daqu.

Baijiu, a prevalent alcoholic beverage, boasts over 2000 aroma compounds, with sulfur-containing compounds being the most influential in shaping its flavor. Benzenemethanethiol, a distinctive odorant in baijiu, is known to enhance the holistic flavor profile of baijiu. Despite its importance, there is very little literature on the biotransformation mechanism of benzenemethanethiol. Thus, extensive research efforts have been made to elucidate the formation mechanism of this compound in order to improve baijiu production. In this study, 12 yeast strains capable of generating benzenemethanethiol were isolated from baijiu daqu, and the Saccharomyces cerevisiae strain J14 was selected for further investigation. The fermentation conditions were optimized, and it was found that the optimal conditions for producing benzenemethanethiol were at 28 °C for 24 h with a 4% (v/v) inoculum of 3.025 g/L L-cysteine. This is the first time that yeast has been shown to produce benzenemethanethiol isolated from the baijiu fermentation system. These findings also suggest that benzenemethanethiol can be metabolized by yeast using L-cysteine and benzaldehyde as precursor substrates.

Effects of Daqu Attributes on Distribution and Assembly Patterns of Microbial Communities and Their Metabolic Function of Artificial Pit Mud.

Daqu provides functional microbiota and various nutrients for artificial pit mud (APM) cultivation. However, little is known about whether its attributes affect the microbiome and metabolome of APM. Here, two types of APM were manufactured by adding fortified Daqu (FD) and conventional Daqu (CD); they were comprehensively compared by polyphasic detection methods after being used for two years. The results showed that FD altered the prokaryotic communities rather than the fungal ones, resulting in increased archaea and Clostridium_sensu_stricto_12 and decreased eubacteria and Lactobacillus. Correlation analysis suggested that these variations in community structure promoted the formation of hexanoic acid, butyric acid, and the corresponding ethyl esters, whereas they inhibited that of lactic acid and ethyl lactate and thus improved the flavor quality of the APM. Notably, pH was the main driving factor for the bacterial community variation, and the total acid mediated the balance between the stochastic and the deterministic processes. Furthermore, the results of the network analysis and PICRUSt2 indicated that FD also enhanced the modularity and robustness of the co-occurrence network and the abundance of enzymes related to hexanoic acid and butyric acid production. Our study highlights the importance of Daqu attributes in APM cultivation, which are of great significance for the production of high-quality strong-flavor Baijiu.

Exploring the response patterns of strong-flavor baijiu brewing microecosystem to fortified Daqu under different pit ages.

The fermentation of strong-flavor baijiu depends largely on the evolution of the microbial community that originated from Daqu and pit mud (PM). Applying fortified Daqu (FD) has been proven to be an effective strategy to improve the quality and yield of baijiu. However, it is unclear what the effects of FD on the liquor brewing microecosystem under different pit ages because of the temporal heterogeneity of the PM community. Taking 2-year (new) and 40-year (aged) pits as the objects, the influence of FD on the metabolic profile, physicochemical parameters, and community diversity in Zaopei and PM was investigated by polyphase detecting approaches. Present results showed that the metabolic profiles of Zaopei were significantly improved by FD, whereas those of PM were mainly dependent on pit age. Aspergillus, Caproiciproducens, and Methanosarcina were more abundant in the aged pit, while Kazachstania, Lactobacillus, and Sphingomonas dominated in the new pit, whether in Zaopei or in PM. The interaction relationships among the communities were also altered by FD, and the co-occurrence network, especially the increased links between archaea and bacteria in the new pit. Notably, this interaction in the aged pit distinctly affected the hexanoic acid content based on the Mantel test. The results of PICRUSt2 analysis inferred that FD perhaps improved the interspecies hydrogen transfer in the new pit and increased the carbon flow of hexanoic acid production during chain elongation in the aged pit. These results provide new insights into the production of high-quality strong-flavor baijiu and the aging of PM.

Thermoflavimicrobium daqui sp. nov., a thermophilic microbe isolated from Moutai-flavour Daqu.

Moutai-flavour Daqu is an important starter to support growth of microorganisms in the fermented process of Moutai-flavour liquor. A novel thermophilic microorganism, designated strain FBKL4.011T, was isolated from Moutai-flavour Daqu samples collected from Guotai distillery in Renhuai, Guizhou province, south-west China. The strain could grow at 45-65 °C (optimum 45 °C). Based on polyphasic analysis, strain FBKL4.011T was affiliated to the genus Thermoflavimicrobium. It formed abundant pale-yellow aerial and substrate mycelium, bearing single endospores (7.0-10.0 µm diameter) on branched long sporophores (5.0 µm diameter). The cell-wall peptidoglycan contained meso-diaminopimelic acid; ribose, glucose and mannose were the primary whole-cell sugars. The major fatty acids were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0. The predominant menaquinone were MK-8 and MK-9. The polar phospholipids contained diphosphatidyl glycerol, phosphatidyl ethanolamine, one unidentified phospholipid and one unidentified lipid. The G+C content of the genome was 43.1 mol%. According to the 16S rRNA gene sequence analysis, strain FBKL4.011T was closely related to Thermoflavimicrobium dichotomicum JCM 9688T (95.3 % sequence similarity), and other members within the family Thermoactinomycetaceae (less than 93.0 % sequence similarity). The DNA-DNA hybridisation data showed low relatedness between strain FBKL4.011T and T. dichotomicum JCM 9688T, Laceyella sacchari KCTC 9790T, Laceyella tengchongensis YIM 10002T, Laceyella sediminis RHA1T(36.7±1.1 %, 30.0±1.2 %, 21.3±2.1 % % and 37.6±0.9 %, respectively). Based on data from the polyphasic analysis, strain FBKL4.011T is considered to represent a novel species of the genus Thermoflavimicrobium, for which the name Thermoflavimicrobium daqui sp. nov. is proposed. The type strain is FBKL4.011T(=KCTC 43036T=CICC 24504T).

Franconibacter daqui sp. nov., a facultatively alkaliphilic species isolated from a Daqu sample.

A novel bacterium, designated strain DL503T, was isolated from a Daqu sample and its taxonomic position determined using a polyphasic taxonomy. Strain DL503T was a Gram-stain-negative, facultatively anaerobic, non-sporulating, motile and coccoid-rod-shaped bacterium. Optimum growth occurred at 20-45 °C, pH 5.0-10.0 and 1.5 % (w/v) NaCl. Comparative analysis of the 16S rRNA gene sequence showed that the isolate belongs to the genus Franconibacter, showing highest levels of similarity with respect to Franconibacter pulveris JCM 16471T (98.94 %) and Franconibacter helveticus DSM 18396T (98.39 %). Cells contained the quinones Q-8 and MK-8, and the polar lipid profile consisted of a mixture of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unidentified polar lipids and three unidentified amino lipids. The DNA G+C content was 53.3 mol% and the major fatty acids were C16 : 0, C17 : 0 cyclo, summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), summed feature 4 (C17 : 1 iso I and/or C17 : 1 anteiso B) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The DNA-DNA relatedness values between strain DL503T and its close relatives, including F. pulveris JCM 16471T and F. helveticus DSM 18396T, were 51.5±0.5 % and 45.2±1.1 %, respectively. Based on phylogenetic analysis, phenotypic and genotypic data, it is concluded that the isolate represents a novel species of the genus Franconibacter, for which the name Franconibacter daqui sp. nov. is proposed. The type strain is DL503T (=LMG 29914T=CGMCC 1.15944T).