The physicochemical properties and microbial community structure of two grades of Nongxiangxing Daqu were analyzed, and the internal relationship between them was investigated. High-throughput sequencing was used to analyze the differences in the microbial community structure between the two grades of Daqu. Meanwhile, we determined their physicochemical properties. Redundancy analysis (RDA) was used to study the relationship between microbes and physicochemical properties. The results showed that the bacterial community structures in the two grades of Daqu were similar, and the dominant bacterial genus were Thermoactinomyces, Weissella, and Lactobacillus, Kroppenstedtia; the domininant fungi were Thermomyces, unclassified_f__Aspergillaceae, and Thermoascus. We found that the relative abundance of Thermoascus in the superior grade was significantly higher than the first grade through differential microbial analysis, while the reverse was observed for unclassified_f__Aspergillaceae. The differential bacteria were all low-abundance species (relative abundance less than 1%). In addition, the moisture content, pH, liquefaction power and saccharification power of the superior grade were significantly lower than the first grade, while the total acid content and esterification power were significantly higher than the first grade. However, there was no significant difference in fermentation power between them. RDA analysis showed that the baterial genera Lactobacillus, Staphylococcus, Weissella and the fungal genus Thermoascus were positively correlated with fermentability and pH, whereas Thermoactinomyces and Kroppenstedtia were negatively correlated with pH. These results may provide a theoretical basis for establishing a more scientific quality evaluation standard for Daqu.

The correlation between the stages of pyrazine synthesis and the microbial flora during the making of Nongxiang Daqu was analyzed, and the effect of microbial interactions on the synthesis of pyrazine during Daqu making was explored. Headspace solid phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) and high-throughput sequencing were used for the analysis of the changes in pyrazine contents and the microbial community structure during Daqu making, respectively. Principal component analysis (PCA) and linear discriminant analysis (LDA) were used for finding out differential microorganisms. The correlation between differential microorganisms and pyrazine metabolites was evaluated using Spearman’s correlation coefficient, and a correlation network between these pyrazine metabolism-related microorganisms and the microbial flora of Nongxiangxing Daqu was constructed. The results showed that nine pyrazine metabolites were detected during Nongxiangxing Daqu fermentation, and there were significant differences in the number and types of pyrazine metabolites between the stages of 1–5 and 7–28 days. The microbial community succession could be divided into two stages: A (1–5 days) and B (7–28 days). At stage A, the differential fungi were mainly Kodamae and Candida, and the differential bacteria were mainly Kosakonia, Pantoea and Methylophilus; at stage B, the differential fungi were mainly Thermoascus and Thermomyces, and the differential bacteria were mainly Thermoactinomyces and Saccharopolyspora. The synthesis of pyrazine metabolites during Nongxiangxing Daqu fermentation was positively correlated Cladosporium and Lichtheimia, but negatively correlated with Thermomyces and Fusarium. These pyrazine metabolism-related microorganisms were positively correlated with most other Nongxiangxing Daqu microorganisms, but showed negative correlation with only a few microorganisms, such as Thermomyces. The results of this study provide a theoretical basis for the analysis of the mechanism of microbial synthesis of pyrazine metabolites in Nongxiangxing Daqu.

This study explored the effect of Bacillus subtilis JP1, isolated from the fermentation process of Nongxiangxing Baijiu, on the assembly and succession of microbial communities in fermented grains (Jiupei). A mixed-culture fermentation system was constructed using Jiupei and yellow water from Nongxiangxing Baijiu production. A bio-perturbation experiment was conducted by adding B. subtilis JP1 to the system. Amplicon sequencing combined with null model analysis was used to investigate the structure, composition and assembly process of microbial communities. R language was employed to calculate the environmental niche breadth of microorganisms to analyze potential reasons for changes in microbial community assembly. The results showed that compared with the control group, B. subtilis JP1 reduced the microbial diversity during fermentation while increasing the relative abundance of ethanol-producing microorganisms. Additionally, B. subtilis JP1 decreased the environmental niche breadth of the microbial community, leading to a significant increase in the number of rare and conditionally rare taxa. The interactions among microorganisms were enhanced, and the complexity of the microbial network was increased. These changes collectively influenced the assembly process of the microbial community during fermentation. The addition of B. subtilis JP1 increased the contribution of heterogeneous selection for bacteria and homogeneous selection for fungi to the microbial community assembly process. Furthermore, it accelerated bacterial succession while slowing down fungal succession. This study elucidates the mechanism of the effect of B. subtilis on the establishment of microbial communities in fermented grains for Nongxiangxing Baijiu, providing a theoretical basis for the targeted regulation of Bacillus during the brewing process of Nongxiangxing Baijiu.

Torulaspora delbrueckii S1, an endophyte isolated from apples, was used in the fermentation of apple vinegar to explore its influence on the flavor of apple vinegar. The volatile flavor substances and organic acids of apple vinegar co-fermented with T. delbrueckii S1 and Saccharomyces cerevisiae (experimental) and that fermented with S. cerevisiae (control) were detected by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) and high performance liquid chromatography (HPLC), respectively, and their differences in flavor substances were compared through biometric analysis. Heatmap cluster analysis showed that T. delbrueckii S1 had no significant effect on the synthesis of volatile substances in apple vinegar, and the apple vinegars could be well distinguished by orthogonal partial least squaresdiscriminant analysis (OPLS-DA). The results of differential metabolites showed that phenethyl acetate, contributing to the typical apple aroma; and phenyl ethanol, benzaldehyde and benzyl alcohol, contributing to floral and fruity aromas, were more prominent in the experimental group, while ethyl caproate, ethyl decanoate and ethyl laurate, responsible for fruit and laurel oil-like aromas, were more prominent in the control group. The results of co-occurrence network analysis showed that T. delbrueckii S1 could enhance the transmissibility of volatile flavor compounds, and consequently increase the complexity of metabolites. In addition, the box plot of organic acids showed that T. delbrueckii S1 could increase the contents of L-lactic acid, L-malic acid and acetic acid. In this study, the effects of apple endophytic fungus T. delbrueckii S1 on the synthesis of flavor compounds in apple vinegar were analyzed, and it provided a basis for further utilization of this strain.

This study aimed to analyze the effect of Issatchenkia orientalis on the microbial community structure and volatile flavor substance synthesis in Nongxiangxing Baijiu Jiupei in order to explore its effect on the microbial interaction and metabolism in the Jiupei. Under laboratory conditions, the microbial system for Nongxiangxing Baijiu brewing consisting of the Jiupei and Huangshui, a by-product of Baijiu brewing, was fermented directly (control group) or after inoculation with a strain of I. orientalis isolated from the Jiupei (experimental group). The composition of microbial communities, microbial interactions, and volatile flavor substances in the 2 groups were analyzed. The results showed that the microbial abundance of Jiupei changed after inoculation with I. orientalis; the relative abundance of Lactobacillus and Issatchenkia increased, and the relative abundance of Paenibacillus and Aspergillus decreased. Moreover, the microbial interaction was strengthened, making the microbial network structure more complex and compact. Compared with the control group, the volatile flavor substances in the experimental group increased significantly, 20 significantly differential volatile flavor substances were identified, and the contents of isopropanol, isoamylol and isobutanol increased significantly. The results of this study disclose the influences of inoculating I. orientalis on the microbial community structure and volatile flavor substances in Nongxiangxing Baijiu Jiupei and help to understand the impact of functional microorganisms on the microbial perturbation and metabolite synthesis in Jiupei.