Evolution of fermented grain yeast communities in strong-flavored baijiu and functional validation of yeasts that produce superior-flavored substances.

BACKGROUND: Baijiu is a well-known alcoholic beverage in China and the quality is determined by various microorganisms during the fermentation process. Yeast is one of the most important microorganisms in the fermentation of baijiu. It has a strong esterification capacity and also affects the aroma. RESULTS: High-throughput sequencing results showed that the fermented grains (jiupei) during baijiu production were mainly composed of eight highly abundant yeast species. The species and abundance of yeasts changed significantly with the fermentation process. The flavor of 30 yeast strains in the jiupei was determined by a sniffing test and gas chromatography-mass spectrometry (GC-MS). The strain with the highest flavor substance content (2.34 mg L-1), named YX3205, was identified as Clavispora lusitaniae. Tolerance results showed that C. lusitaniae YX3205 can tolerate up to 15% (v v-1) ethanol. In a solid-state simulated fermentation experiment, the content of 24 flavor substances was significantly increased in the fortified group, and the total ester content reached 4240.73 µg kg-1, which was 2.8 times higher than that of the control group. CONCLUSION: The present study demonstrated the potential of C. lusitaniae YX3205 to enhance the flavor of baijiu, thereby serving as a valuable strain for the improvement of the flavor quality of baijiu. © 2024 Society of Chemical Industry.

Spatial heterogeneity of microbiota and flavor across different rounds of sauce-flavor baijiu in Northern China.

Sauce-flavor baijiu (SFB) is a traditional Chinese distilled liquor crafted through a distinctive brewing process, involving seven rounds of stack fermentation (SF) and pit fermentation (PF). To date, there remains a knowledge gap regarding the microbial succession and flavor throughout all rounds of SFB with distinctive northern characteristics. Through LEfSe analysis, Saccharopolyspora, Virgibacillus, Thermoascus and Thermomyces, and Lactobacillus and Issatchenkia were found to be the most differentially representative genera in SF and PF, respectively. A total of 93 volatile flavor compounds were found in base baijius through the gas-chromatography mass spectrometry. Moreover, 29 volatile flavor substances with significant difference in base baijius of different rounds were revealed using the OPLS-DA model and VIP values and Spearman correlation analysis shows that bacteria have a greater impact on differential flavor compounds than fungi. This study provides a new perspective and insight into the brewing of northern SFB.

Comparison of fungal communities and flavour substances in surface and inner layers of fermented grains during stacking fermentation of sauce-flavour baijiu.

The stacking fermentation process plays a vital role in the production of sauce-flavour baijiu. The aim of this paper is to elucidate the effects of environmental variables on the fungal communities of different layers of fermented grains (zaopei) during the sixth round of stacking and the changes in volatile flavour substances during this process. The composition of the fungal communities in different layers during the stacking fermentation process was analysed. Principal coordinate analysis (pCoA) showed that the dominant fungal communities in different layers differed significantly with the stacking fermentation process. The dominant fungal genera were Thermoascus, Thermomyces and Issatchenkia. The total content of flavouring substances in the surface layer of zaopei were higher, but the types of flavouring substances were less than in the inner layer. The relationship between temperature, moisture content, acidity, starch content and reducing sugar content and microbial community was analysed by Redundancy analysis. The results showed that the correlation between microbial communities and physicochemical indexes in different layers of zaopei varied. The core fungal genera in the surface layer were mainly influenced by acidity, and the microorganisms in the inner layer were more strongly correlated with temperature. Spearman correlation coefficient revealed the correlation between fungal community and volatile flavour substances, and the results showed that microorganisms in different layers of zaopei have different correlations with flavour substances. This study contributes to the understanding of the evolution of different layers fungal communities during the stacking of sauce-flavour baijiu and their relationship with volatile aroma substances.

Characteristics and Correlation of the Microbial Communities and Flavor Compounds during the First Three Rounds of Fermentation in Chinese Sauce-Flavor Baijiu.

Sauce-flavor Baijiu is representative of solid-state fermented Baijiu. It is significant to deeply reveal the dynamic changes of microorganisms in the manufacturing process and their impact on the formation of flavor chemicals correlated with the quality of Baijiu. Sauce-flavor Baijiu manufacturing process can be divided into seven rounds, from which seven kinds of base Baijius are produced. The quality of base Baijiu in the third round is significantly better than that in the first and second rounds, but the mystery behind the phenomenon has not yet been revealed. Based on high-throughput sequencing and flavor analysis of fermented grains, and correlation analysis, the concentrations of flavor chemicals in the third round of fermented grains were enhanced, including esters hexanoic acid, ethyl ester; octanoic acid, ethyl ester; decanoic acid, ethyl ester; dodecanoic acid, ethyl ester; phenylacetic acid, ethyl ester; 3-(methylthio)-propionic acid ethyl ester; acetic acid, phenylethyl ester; hexanoic acid, butyl ester, and other flavor chemicals closely related to the flavor of sauce-flavor Baijiu, such as tetramethylpyrazine. The changes in flavor chemicals should be an important reason for the quality improvement of the third round of base Baijiu. Correlation analysis showed that ester synthesis was promoted by the bacteria genus Lactobacillus and many low abundances of fungal genera, and these low abundances of fungal genera also had important contributions to the production of tetramethylpyrazine. Meanwhile, the degrading metabolic pathway of tetramethylpyrazine was investigated, and the possible microorganisms were correlated. These results clarified the base Baijiu quality improvement of the third round and helped to provide a theoretical basis for improving base Baijiu quality.

Clostridium btbubcensis BJN0001, a potentially new species isolated from the cellar mud of Chinese strong-flavor baijiu, produces ethanol, acetic acid and butyric acid from glucose.

A novel strain, designated BJN0001, was isolated from the cellar mud of Chinese strong-flavor baijiu. The complete genome of strain BJN0001 was 2,688,791 bp and annotated with 2610 genes. Whole-genome similarity metrics such as average nucleotide identity (ANI) of BJN0001 with reference genomes reveals clear species boundaries of < 95% ANI value for species. The DNA-DNA hybridization (DDH) values of BJN0001 with the type species were all lower than 70% DDH value for species. Based on these results, the strain BJN0001 was considered a potentially new species of the genus Clostridium. Meanwhile, the fermentation characteristics indicated that the strain had the capability to convert glucose to ethanol, acetic acid and butyric acid, which could provide basic data for revealing its function in baijiu fermentation. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03271-7.

Sedimentation Models and Development Mechanisms of Organic-Rich Shales of the Lower Carboniferous Dawuba Formation: A Case Study in the Yaziluo Rift Trough, South of Guizhou Province, Southern China.

China has made a breakthrough in shale gas production in the deepwater shelf shales of the Lower Cambrian Qiongzhusi Formation and the Upper Ordovician-Early Silurian Wufeng-Longmaxi Formation. In recent years, active shale oil and gas shows have also been found in the shale system of the Lower Carboniferous Dawuba Formation in the Yaziluo rift trough, south of Guizhou province in Southern China, which was formed in the tensional geotectonic setting of the Palaeo-Tethys Ocean from the Devonian through the Carboniferous to the Permian. This tectonic background makes the sedimentary environments and organic matter accumulation mechanisms of Dawuba shales vastly different from deepwater shales. To better understand the deposition and organic matter accumulation mechanisms of marine shale developed in the rift trough, we carried out detailed field surveys and drilling data interpretation to study the lithological assemblage, organic matter, and elemental geochemical characteristics of Dawuba shales. The results show the following: (1) The study area is located in a platform-slope-basin depositional model like the Florida-Bahama platform-trough system of the west Atlantic margin, with a rapidly geomorphologic variation from basin to bank, dominated by a coastal sandstone and mudstone system in the northwest, a marlite and mudstone slope system around the rift trench (Liupanshui county), and a deep water fine-grained-turbidite system in the southeast (Ziyun county). (2) Major element (ME), trace element (TE), and rare earth element (REE) data indicate significant terrestrial source material input [total organic carbon (TOC) correlates well with Ti/Al], high deposition rates [mean (La/Yb)N of 1.41], and complex oxic-dysoxic conditions (U/Th mainly between 0 and 0.5), which illustrate substantial terrigenous sedimentary input and changes in redox conditions in deep water. (3) The input of organic matter from terrestrial sources in the study area is predominant compared with internal basin-originated organic matter, and the organic matter type is mainly Type II2 or Type III. Stable carbon isotope (δ13Ccarb) data of carbonate rocks also indicates that the widely developed upwelling in this region brings abundant nutrients, which also contributes to organic matter enrichment. Organic-rich shales exist in the Yaziluo rift trough under the influence of strong tensile action. The results of the study are essential for understanding the sedimentology and hydrocarbon exploration in similar rift trough areas within the Paleo-Tethys Ocean.

Main Controlling Factors and Models of Organic Matter Accumulation in Lower Carboniferous Dawuba Formation Shale in Southern Guizhou, China.

A set of high-quality marine facies organic-rich shales developed in the Lower Carboniferous Dawuba Formation, which is considered to be the main target of shale gas exploration and development in Guizhou Province. In this paper, 53 samples from Well ZY1 are selected, and the core observation data, field-emission scanning electron microscopy (FE-SEM) images, and geochemical data of these samples are analyzed. On the basis of these data, the main influencing factors of organic matter enrichment in the Dawuba Formation shale were identified and an organic matter accumulation model was established. The results show that total organic carbon (TOC) values of the Dawuba Formation in the ZY1 well vary between 1.97 and 4.11%, with high values appearing at the depths of 2796-2814 m (3.00-4.11) and 2877-2894 m (1.97-3.49). The redox-sensitive element enrichments are generally low, indicating that these samples were deposited under oxic-suboxic conditions. The micronutrients (Zn, Cu, and Ni), biological Ba (BaXS), and P/Al also show low values, indicating low primary productivity. The chemical index of alteration (CIA) and terrigenous clastic input index (Ti/Al) showed two obvious high-value zones, indicating that shale in the study area was affected by terrigenous inputs. Similarly, the calculation results show that Fe/Mn and Rb/K values have two abnormal data segments at the same depth. The anomaly of these data at the same depth section further suggests that the shale was affected by terrigenous input during deposition. Moreover, the terrigenous input reaches the maximum in the above TOC high-value region, and it is inferred by combining with the core observation results that the gravity flow occurs in this depth. The carbon isotope of kerogen (δ13Corg) ranges from -26.84 to -24.36%, indicating that the source of organic matter is likely to be terrestrial plants. This is further supported by the widespread presence of filamentous organic matter using FE-SEM, despite the low productivity and poor preservation conditions during deposition of the Dawuba Formation; the enhanced terrigenous input may have provided additional sources of organic matter for the Dawuba shale.

Correlation between microbial communities and flavor compounds during the fifth and sixth rounds of sauce-flavor baijiu fermentation.

Sauce-flavor baijiu is a representative of Chinese traditional fermented baijiu using grains as the raw materials through the co-fermentation of microorganisms. The whole manufacturing process includes 7 times of distillation and generates 7 kinds of base baijius. The final product is a mixture of the 7 kinds of base baijius. Thus the base baijius greatly affect the quality of the final product. The quality of the base baijiu obtained by the sixth distillation is obviously poorer than that of the fifth one. However, the reason is still unclear and limits the quality control of baijiu fermentation. In this study, the flavor substances and microbiota in the up, middle and bottom layers of fermented grains in the fifth and sixth rounds were compared. Some flavor esters showed obviously decreased concentrations in the sixth round, including ethyl benzoneacetic acid, ethyl hexanoic acid, ethyl dodecanoic acid, diethyl butanedioic acid, and ethyl 2-hydroxyl-propanoic acid. Meanwhile, an off-flavor p-cresol was detected in the sixth round. Correlation analysis of flavor chemicals and microbiota indicated that fungi in the fifth round played an important role for ester synthesis. Some bacterial and fungal species were both positively correlated with p-cresol synthesis, and the related p-cresol metabolic pathways were proposed for the first time. These results revealed flavor divergences of fermented grains between the fifth and sixth rounds, and will ultimately help to improve baijiu quality.