Characterizing the regulation mechanism of microbial community and metabolites in multi-layer vinegar starter ( <em>Cuqu</em>) via <em>Muqu</em> fortification strategy

Improving the quality of <em>Cuqu</em> is one of the necessary strategies increasing the quality and yield of fresh vinegar. Enhancing the consistency and characteristics of <em>Cuqu</em> is currently a prominent area of research. Here, we investigated the impact of fortified <em>Muqu</em> (FM) consisting of <em>Bacillus velezensis</em> and <em>Bacillus subtilis</em> on enhancing the quality of <em>Cuqu</em> compared to conventional <em>Muqu</em> (CM). Our results demonstrated that FM significantly increased the abundance of <em>Bacillus</em> by 1.85 to 8.19 times, as well as substantially elevated <em>Kroppenstedtia</em> abundance in <em>Cuqu</em>. Source Tracker analysis revealed a higher proportion of bacteria originating from FM in <em>Cuqu</em> 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 <em>Cuqu</em> 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 <em>Cuqu</em>. These results elucidated the regulatory function of FM in forming microbial communities and metabolic mechanisms within <em>Cuqu</em>, providing valuable insights for optimizing and predicting traditional fermentation processes in industrial production.