Improving the quality of Cuqu is one of the necessary strategies increasing the quality and yield of fresh vinegar. Enhancing the consistency and characteristics of Cuqu is currently a prominent area of research. Here, we investigated the impact of fortified Muqu (FM) consisting of Bacillus velezensis and Bacillus subtilis on enhancing the quality of Cuqu compared to conventional Muqu (CM). Our results demonstrated that FM significantly increased the abundance of Bacillus by 1.85–8.19 times, as well as substantially elevated Kroppenstedtia abundance in Cuqu. Source Tracker analysis revealed a higher proportion of bacteria originating from FM in Cuqu compared to those derived from CM, indicating superior environmental adaptability for bacteria in the FM. Additionally, upregulation in expression levels of pyrazine-related key enzymes within Cuqu microbial communities was observed due to FM, resulting in a remarkable 39.70-fold increase in pyrazine compounds production. Multiphase detection technology confirmed that FM improved similarity in microbial community structure and function at different spatial positions within Cuqu. These results elucidated the regulatory function of FM in forming microbial communities and metabolic mechanisms within Cuqu, providing valuable insights for optimizing and predicting traditional fermentation processes in industrial production.

To uncover the origin of the microbial community in strong-flavor Daqu, this study investigated the traceability of the microbial communities in Daqu and environmental samples from old and new production bases of one distillery. The production environment was thought to play a key role in the manufacturing process of Daqu. The results of SourceTracker showed that the major microbial sources of the raw starter brick before incubation from the old base were the raw materials and the production tools, and the environment in the old base contributed more to the bacterial communities in the raw starter brick and mature Daqu (Daqu just transferred to the storeroom). However, the fungal community was relatively stable and less affected by the environmental microbiota. As powdered aged Daqu (above three months old) was used to improve the quality of fresh Daqu, the microbial sources of the raw starter brick and mature Daqu from the new production base were the aged Daqu. A suitable environment for the production of Daqu could be created by proper fortification methods, which is of great significance to maintain the quality of Daqu.

The effect of spatial heterogeneity on the microbial community and physicochemical properties during the primary fermentation of Daqu were investigated by high-throughput sequencing technology and conventional detection methods. Nongxiangxing baijiu Daqu inoculated with the unique ripe starter obtained by gradually culturing and expanding Daqu treated by cosmic rays was used. The results showed that although the intensity of change in driving factors varied among layer, their trends were the same. The liquefying, saccharifying and esterifying power of Daqu were higher in the bottom layer than in the upper and middle layers at the same fermentation time and the fluctuation was small. The microbial community of Daqu was composed of 12 dominant bacterial genera, including Lactobacillus, Weissella, Bacillus, Kosakonia, Staphylococcu and Thermoactinomyces, and seven dominant fungal genera, such as Pichia, Thermoascus, and Rhizomucor. Principal co-ordinates analysis and hierarchical clustering analysis showed significant differences in the bacterial and fungal community structure among fermentation stages and layers. Procrustes analysis and Mantel test showed that moisture had a significant effect on the bacterial community in Daqu, and acidity had a significant effect on the bacterial community in the middle and bottom layers of Daqu. Moreover, moisture had a significant effect on the fungal community in the upper and middle layers of Daqu. Redundancy analysis showed that moisture and acidity were positively correlated with Lactobacillus and Pichia, while driving factors had different influences on the microbial communities in different layers of Daqu. Therefore, the interaction and co-occurrence patterns of microbial genera in Daqu could change due to the differences in driving factors among different layers of Daqu. These results suggested that regulating driving factors during the Daqu making process is an effective way to improve the microbial community structure and quality of Daqu.

In this study, a small-scale simulated fermentation system of fermented grains (Jiupei in Chinese) without pit mud (PM) was used as a control, and the differences in the microbial community and metabolite components between the PM and control groups were compared. The results indicated that PM significantly enhanced the contents of major flavor compounds in Jiupei including butyric acid, caproic acid, ethyl acetate, ethyl butyrate, and ethyl caproate, and reduced the contents of lactic acid and ethyl lactate. For both groups, the relative abundance of Lactobacillus, one of the dominant bacteria in the upper and bottom layers of Jiupei, significantly increased at the initial stage, and showed a slight difference between them at the end of fermentation. The decay period of Lactobacillus in the PM group was about 15 days earlier than that in the control group. At the same time, the relative abundance of Rhodococcus in the control group, and the relative abundance of Kroppenstedtia, Clostridium_sensu_stricto_12, and Acetobacter in the PM group increased. The abundance of most of the enzymes in the Embden-Meyerhof-Parnas (EMP) pathway, as well as that of caproic acid synthase (EC 1.3.1.38, EC 2.3.1.16, EC 6.2.1.1) and butyric acid synthase (EC 2.3.1.9, EC 2.8.3.8) increased during fermentation, and the abundance of these enzymes was significantly higher in the bottom layer of Jiupei than in the upper layer. The abundance of lactate dehydrogenase (EC 1.1.1.27), which uses lactic acid as a substrate, increased in the PM group. These findings reveal the contribution of PM to the microbial communities and metabolite components of Jiupei.

The quality and aroma of strong-flavor Baijiu are mainly dependent on Daqu, pit mud (PM), and the interaction of both. However, little is known about how their combination patterns affect the microbiome and metabolome of Zaopei, especially the metabolic function of rare taxa. Here, an experiment on industrial size was designed to assess the effects of 6 combinations (3 kinds of Daqu × 2 kinds of PM) on the composition and assembly of different taxa, as well as the flavor profile. The results showed that Zaopei’s microbiota was composed of a few abundant taxa and enormous rare taxa, and rare bacterial and abundant fungal subcommunities were significantly affected by combination patterns. The assembly processes of abundant/rare taxa and bacterial/fungal communities were distinct, and environmental changes mediated the balance between stochastic and deterministic processes in rare bacteria assembly. Furthermore, specific combination patterns improved the f lavor quality of Zaopei by enhancing the interspecies interaction, which was closely related to rare taxa, especially rare bacteria. These findings highlighted that rare bacteria might be the keystone in involving community interaction and maintaining metabolic function, which provided a scientific foundation for better understanding and regulating the brewing microbiota from the viewpoint of microbial ecology.