Chinese strong-flavor liquor, a distinctive category of Chinese spirits, contained over 1,300 types of flavor compounds and was characterized by its harmonious taste and cellar aroma. Highly favored by Chinese consumers, the production and sales of Chinese strong-flavor liquor constituted approximately 70% of the total output of China’s liquor industry (He et al., 2020). Daqu is a kind of starter culture used in liquor making, which was mainly used for solid state fermented liquor. The Daqu fermentation process generated a diverse array of aromatic compounds, which endowed the liquor with its distinctive aroma and flavor profile (Wang et al., 2017a). Daqu is usually classified into three types, depending on the maximum temperature reached during the fermentation process, i.e., low temperature Daqu (LT-Daqu) (45–50°C), medium temperature Daqu (MT-Daqu) (50–60°C), and high temperature Daqu (HT-Daqu) (60–65°C) (Wang et al., 2011). Meanwhile, MT-Daqu, the most widely used starter in the production of traditional Chinese strong-flavor liquor, is rich in bacteria, fungi and enzymatic systems and serves as both the foundation of Chinese strong-flavor liquor production and the key determinant of its quality (Fan et al., 2007; Zhang et al., 2009).

During the Daqu production process, sophisticated techniques of natural inoculation and open fermentation were employed. These methods adeptly harnessed the microbial diversity of the brewing environment, primarily bacteria and fungi (Du et al., 2019), thereby imparting Daqu with its unique flavor and superior quality. Bacteria served as the crucial microbial group in the fermentation process of Daqu that produced protease and flavoring substances (Wang et al., 2020). In recent years, the rapid advancement of high-throughput sequencing technology had led to its widespread application in analyzing the microbial community structures across diverse environments (Bruno et al., 2016; Fan et al., 2020; Wang et al., 2017b). The Chao index (representing richness), Shannon and Simpson index (representing diversity), and coverage index (representing coverage) were used to describe the microbial composition of Daqu (Zhuansun et al., 2022). It has been reported that Firmicutes and Proteobacteria were the dominant bacterial phyla in the fermentation process of Daqu (He et al., 2019; Hu et al., 2017; Sun et al., 2016; Tao et al., 2014; Wang et al., 2017b). The dominant bacterial genera were mainly Lactobacillus and Bacillus (Ge et al., 2024; Tang et al., 2023). In addition, research on bacterial community has primarily focused on the interactions among bacterial communities and the correlations between bacterial communities and physicochemical properties, etc. (Qian et al., 2021; Zhang et al., 2020; Zhi et al., 2016). These studies not only enhanced our understanding of Daqu’s microbial ecology but also offered a scientific foundation for optimizing the Daqu fermentation process. However, studies on the succession characteristics and mechanisms of Daqu fungal community are still limited.

Fungal communities play a crucial role in the fermentation process of Daqu (Zheng et al., 2012), such as producing alcohol, enzymes, and flavor compounds (Wang et al., 2017b), and providing the necessary saccharification, liquefaction, and proteolytic capabilities required by Daqu. Moreover, they also produce a range of organic acids and other important substances, generating various flavor components, and enhancing the quality of Daqu (Wang et al., 2017c). Currently, research on the fungal communities in Daqu fermentation paralleled that of bacterial communities, focusing primarily on the shifts in fungal communities composition, the interactions between bacterial and fungal communities, the impact of the fungal communities on physicochemical properties, and the influence of physicochemical factors on the fungal communities (Li et al., 2019a; Safatov et al., 2010; Zhang et al., 2007). Much of the published literature relies on Redundancy analysis (RDA) to elucidate correlations between microbial community structure and physicochemical factors (Jin et al., 2019; Zhu et al., 2022a), however, the identification of keystone species in fungal communities and the keystone drivers influencing them remain unclear. Consequently, the identification of keystone fungal species may elucidate the assembly mechanisms of the Daqu microbial community, thereby offering technical support for enhancing Daqu quality and for research into its functional roles and enhancement strategies. Moreover, the response and adaptability of the keystone fungal species to environmental physicochemical factors may also have been a critical determinant in the Daqu fermentation process.

Therefore, this study analyzed the dynamic changes in fungal community composition at various time points throughout the MT-Daqu fermentation process (0, 10, 20, 30, and 120 days). The study aimed to explore (i) changes in diversity and composition of fungal community during Daqu fermentation, (ii) interaction and keystone species composition of Daqu fungal community, and (iii) key drivers of keystone species of Daqu fungal community.

Understanding the succession patterns and interaction dynamics of the Daqu fungal community is crucial for the production of Chinese strong-flavor liquor. Although the composition and functionality of numerous Daqu fungal community have been examined, research into the keystone species and their specific roles has been somewhat constrained. Therefore, a systematic analysis of the role and function of the keystone species within the Daqu fungal community is essential for optimizing the brewing process of Chinese strong-flavor liquor.

Our initial study findings predominantly disclosed a pronounced disparity in fungal diversity between the initial sampling on day 10 and subsequent samplings. Notably, the fungal communities during the pre-fermentation stage (day 0) were significantly different from those observed during the fermentation phase (from day 0 to day 30) and the storage phase (from day 30 to day 120). Ascomycota emerged as the predominant fungal phylum in the fermentation process of Daqu, aligning with previous research findings (Dong et al., 2024; Tong et al., 2023; Zhou et al., 2023). Additionally, a pronounced shift in the microbial community structure was observed between days 10 and 20 of the fermentation process. The relative abundance of Saccharomycopsis increased progressively throughout the fermentation process, ultimately becoming the predominant strain within the Daqu fungal community. Saccharomycopsis, a prevalent microbial flora in Daqu, is capable of producing α-amylase and glucoamylase, thereby significantly enhancing the glycosylation capacity of the mixture (Chen et al., 2021; Li et al., 2019a; Xie et al., 2021).

A notable secondary finding of our study was that the synergistic interactions among keystone taxa within the Daqu fungal community are crucial for maintaining its stability. Network hubs, identified as potential keystone taxa, play a pivotal role in the stability of community structure and function (Banerjee et al., 2019; Delgado-Baquerizo et al., 2016; Xiong et al., 2020). Employing co-occurrence network analysis in this study, we identified keystone species within the fungal fermentation process. Unidentified fungi, along with Udeniomyces pyricola, Aspergillus penicillioides, Udeniomyces megalosorus, Arachnomyces jinanicus and Alternaria sp., were revealed as keystone taxa that play a critical role in the dynamics of the fungal community. Although specific contributions of Udeniomyces pyricola and Udeniomyces megalosorus to the Daqu fermentation process are not explicitly detailed in the literature, their classification within the yeast genus suggests potential synergistic interactions with yeasts in analogous environments, potentially contributing to liquor fermentation and the development of distinctive flavors. It is plausible that these species may have influenced the fermentation process and flavor profile. Future research may provide more detailed insights into the role of these yeasts in Daqu. Concurrently, the specific role of Arachnomyces jinanicus warrants further elucidation. We hypothesize that Arachnomyces jinanicus may secrete a range of enzymes involved in the metabolic pathways within the Daqu fermentation environment, potentially influencing the synthesis of flavor compounds and thus impacting the taste and aroma profiles of the liquor product. Aspergillus penicillioides, capable of secreting a diverse array of hydrolytic enzymes including α-amylase, glycosylase, glucosidase, endoglucanase, and β-glucosidase, promotes the succession of the Daqu microbial community and the fermentation of fermented grains (Li et al., 2019b; Xia et al., 2022). Alternaria sp., a keystone functional fungal genus within the wheat microbiota, plays a significant role in various metabolic pathways during the early stages of incubation. Finally, the unidentified fungi may also have held a unique role in the ecosystem, and additional research might have been warranted to explore and ascertain their specific ecological and economic significance. In conclusion, keystone species within the Daqu fermentation process likely influenced the assembly, succession, stability, and adaptability of the entire microbial community through robust biological interactions. These interactions ultimately shaped the flavor and aroma profiles of Chinese strong-flavor liquor.

Moreover, given the paramount importance of physicochemical factors in the fermentation process of liquor, precise management and modulation of Daqu fungal community were essential during the initial phase of fermentation (Azzolini et al., 2012; Lu et al., 2015; He et al., 2022). As the microbial succession progressed, the role of deterministic selection became increasingly significant (Dini-Andreote et al., 2015), and the keystone species within the Daqu ecosystem under specific conditions were found to exhibit a correlation with their environmental determinants (Mao et al., 2022; Wang et al., 2021). During the fermentation process, the keystone microbial strains were primarily influenced by the levels of moisture and acidity. Moisture, acting as a vital energy substrate for the microbial community, was essential for catalyzing a range of biochemical reactions within the Daqu matrix (Ma et al., 2021). These processes included starch hydrolysis, protein degradation and esterification processes, which were fundamental to the biosynthesis of the key flavor compounds that imparted the distinctive characteristics to the distilled spirit. In addition, the acidity exerted a significant impact on the growth and metabolic activities of microorganisms within the fermentation medium. An optimal acidic environment was not only instrumental in fostering the proliferation and reproduction of beneficial microorganisms, including bacteria and yeast, which contributed to the establishment of a dominant microbiota, but it also served to suppress the proliferation of detrimental microorganisms. The meticulous regulation of the acid generation range throughout the fermentation process was crucial for ensuring the smooth progression of Chinese strong-flavor liquor fermentation (Zhu et al., 2022b).

This study conducted a comprehensive analysis of the composition and diversity within the Daqu fungal community, with a focus on identifying keystone species and their pivotal roles. The research revealed that moisture and acidity were the primary environmental determinants influencing these keystone species. The findings systematically elucidated the succession patterns and underlying mechanisms of fungal communities during the Daqu fermentation process, enhancing our understanding of the keystone species’ critical function in maintaining community stability and their adaptive responses to environmental changes. Consequently, this study offered novel insights into the intricate dynamics of fungal interactions in the production of Chinese strong-flavor liquor and served as a valuable reference for the future optimization and selection of Daqu fungal community.

However, there were several limitations that might need to be addressed in our future work. First, the use of amplicon-based sequencing, as opposed to metagenomics, constrains the study’s capacity to fully capture the functional potential of the fungal community. Second, a larger sample size is needed to enhance statistical power and reduce the likelihood of false positives. In addition, in order to better understand the driving effects of keystone taxa and physicochemical factors on fungal community succession during Daqu fermentation, it is necessary to expand the sampling range of Daqu samples and carry out a comprehensive tracking and monitoring project on fungal community and Daqu ecosystem characteristics.